Mapkap kinase-2 as a specific target for blocking proliferation of P53-defective cells

ABSTRACT

The present invention relates to compounds and pharmaceutical compositions for treating cellular proliferative disorders, e.g., in patients having one or more p53-deficient cells, screening assays for identifying such compounds, and methods for treating such disorders.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation-in-part of U.S. application Ser. No.11/273,567, filed Nov. 14, 2005, which claims benefit of U.S.Provisional Application No. 60/627,352, filed Nov. 12, 2004, each ofwhich is hereby incorporated by reference.

This application also claims benefit of U.S. Provisional ApplicationNos. 60/794,451, filed Apr. 24, 2006; 60/800,298, filed May 12, 2006;and 60/873,904, filed Dec. 8, 2006, each of which is hereby incorporatedby reference.

STATEMENT AS TO FEDERALLY SPONSORED RESEARCH

The present research was supported by grants from the NationalInstitutes of Health (grant numbers GM60594 and CA112967) and from theNational Institute of Environmental Health Sciences (grant numberES015339). The U.S. government has certain rights to this invention.

BACKGROUND OF THE INVENTION

The maintenance of genomic integrity is essential for the health ofmulti-cellular organisms. DNA damage checkpoints constitute a mechanismwhere cell division is delayed to allow repair of damaged DNA, or if theextent of DNA damage is beyond repair, induce apoptosis. The three majorDNA damage-responsive cell cycle checkpoints are the G₁/S checkpoint,intra S-phase checkpoint, and the G₂/M checkpoint.

In response to DNA damage, eukaryotic cells activate a complex signalingnetwork to arrest the cell cycle and facilitate DNA repair. Thissignaling network has traditionally been divided into two major proteinkinase pathways, one mediated by Ataxia-Telangiectasia mutated (ATM)through Chk2, and the other mediated by Ataxia-Telangiectasia and Rad-3related (ATR) through Chk1. Some cross-talk exists between the ATM/Chk2and ATR/Chk1 kinase pathways, particularly when signaling through onepathway is partially or totally deficient. Normally, however thepathways show only partial functional overlap in response to particularforms of DNA damage. The ATM/Chk2 pathway responds primarily to DNAdouble strand breaks (DSBs), while the ATR/Chk1 pathway is activated bybulky DNA lesions, and following replication fork collapse duringS-phase. The tumor suppressor protein p53 is a major downstream effectorof these DNA damage kinase pathways. In normal cells, p53-dependentsignaling results in G₁ arrest, mainly mediated by transcriptionalupregulation of p21. In addition, p21 also appears to play a role insustaining the G₂ checkpoint after γ-irradiation. If the DNA damage isextensive, however, then p53-dependent pathways target the damaged cellfor apoptotic cell death through both the intrinsic and extrinsicpathways. Most tumor cells show specific disruptions in the p53 pathway,leading to selective loss of the G₁ checkpoint. These cells are thenentirely dependent on intra-S and G₂/M checkpoints to maintain theirgenomic integrity in response to DNA damage.

In contrast to the DNA damage-specific activation of Chk1 and Chk2, thep38MAPK pathway is a general stress-activated kinase pathway thatresponds to various cellular stimuli, including cytokines,hyperosmolarity, and UV irradiation. Activity of p38MAPK is importantfor G₂/M checkpoint function in immortalized fibroblasts and HeLa cellsfollowing UV exposure. Furthermore, MAPKAP Kinase-2 (MK2) is thecritical downstream effector kinase of p38MAPK required for UV-inducedcell cycle checkpoints in U2OS cells.

Whether the observed activation of p38 MAPK/MK2 is a direct result ofUV-induced DNA lesions, or results instead from other non-genotoxiceffects of UV radiation has been unclear. Similarly, whether thep38MAPK/MK2 pathway is an important part of a general cellular responseto genotoxic stress has been unclear. There exists a need to betterunderstand this checkpoint and to develop methods and therapies fordisease treatment based on this improved understanding.

SUMMARY OF THE INVENTION

We now report that MAPKAP kinase-2 is specifically activated in responseto DNA damage caused by chemotherapeutic agents in an ATR and/orATM-dependent manner, and that MAPKAP kinase-2 is critical for theactivation of G₁, S-phase and G₂/M checkpoints after exposure to thesedrugs. Down-regulation of MAPKAP kinase-2 using RNA interferenceprofoundly increases the anti-proliferative and cytotoxic effects ofcisplatin and doxorubucin on tumor cells in vitro, and in a murine tumormodel in vivo. MAPKAP kinase-2 depletion is especially effective inincreasing the chemosensitivity of p53-deficient cells, suggesting thatcompounds that target MAPKAP kinase-2 can be used as specifictherapeutics that can sensitize p53-deficient tumor cells withoutsensitizing normal cells. At the systems level, in response to DNAdamage, Chk1 and MAPKAP kinase-2 appear to function in parallelindependent pathways that converge to phosphorylate similar moleculartargets, such that checkpoint abrogation following MAPKAP kinase-2depletion can be rescued by overexpression of Chk1.

Based on these results, we have invented novel methods of treatingcellular proliferative disorders by inhibiting MAPKAP kinase-2expression. We have also discovered MAPKAP kinase-2 inhibitors,pharmaceutical compounds containing such inhibitors that are useful fortreating cellular proliferative disorders, and screening methods foridentifying additional inhibitors. The methods and compounds of theinvention may be used, for example, to treat cancer or to aid in thedevelopment of other anti-cancer therapies.

Accordingly, in one aspect, the invention features a method for treatinga cellular proliferative disorder in a patient that includes the stepsof: (a) determining whether the patient has a p53-deficient cell; and(b) if the patient has a p53-deficient cell, administering to thepatient a compound, e.g., UCN-01, that is capable of inhibiting anactivity of a MAPKAP kinase-2 polypeptide. Any method can be used todetermine whether the patient has a p53-deficient cell, e.g., anantibody assay of a cell sample, e.g., from a tumor biopsy. The MAPKAPkinase-2 inhibition can be either specific or non-specific. The activitybeing inhibited may include, for example, MAPKAP kinase-2 polypeptideexpression or substrate-binding. The method may also include the step ofadministering an additional treatment to the patient, such as achemotherapeutic agent or radiation therapy, such that the compound andthe chemotherapeutic agent or the radiation therapy are administered inamounts sufficient to treat the patient's cellular proliferativedisorder. The additional treatment may be administered simultaneously ornonsimultaneously, e.g., up to twenty-eight days apart, in relation tothe administration of the inhibitory compound. Any chemotherapeuticagent or radiation therapy known in the art may be useful in the methodsof the invention. Exemplary chemotherapeutic agents are antimicrotubuledrugs, e.g., nocodazole; compounds that create double-strand DNAbreakage, e.g., doxorubicin and daunorubicin; compounds that inducesingle-strand DNA breaks, e.g., camptothecin; and cross-linking agents,e.g., cisplatin. Exemplary cellular proliferative disorders includeneoplasms, e.g., any known form of cancer. In one embodiment, a solidtumor may be treated by injecting a MAPKAP kinase-2 inhibitor, alone orin combination with an additional therapeutic agent, directly into thetumor or by systemic administration. If given as a monotherapy, thecompound is administered in an amount sufficient to treat the patient'scellular proliferative disorder; alternatively, in the case ofcombination therapy, the combination of compounds is collectivelyadministered in an amount sufficient to treat the patient's cellularproliferative disorder.

An inhibitory compound used in the foregoing method may include acovalently-linked moiety capable of translocating across a biologicalmembrane, such as a penetratin or TAT peptide. Alternatively, such acompound may be administered in the form of a prodrug. Suitablecompounds include small molecule inhibitors of MAPKAP kinase-2biological activity, RNA molecules useful in RNA interference therapy,RNA molecules useful in antisense therapy, and peptides capable ofinhibiting a MAPKAP kinase-2 polypeptide. For example, an RNA moleculeuseful in the methods of the invention includes a double-stranded smallinterfering nucleic acid (siNA) molecule that is capable of directingcleavage of a MAPKAP kinase-2 RNA via RNA interference, wherein eachstrand of the siNA molecule is about 18 to 23 nucleotides in length, andone strand of the siNA molecule includes a nucleotide sequence that issubstantially identical to the sequence of the MAPKAP kinase-2 RNA. Inone embodiment, the siNA molecule includes RNA, the sequence of such RNAincluding, for example, any one of SEQ ID NOs: 29-32. Small hairpinnucleic acid (shNA) molecules may also be used in the methods of theinvention. Alternatively, antisense therapy may be performed byadministering a nucleobase oligomer, wherein the sequence of theoligomer is complementary to at least 10 consecutive residues of anucleotide sequence encoding a MAPKAP kinase-2 polypeptide. Therapy mayalso be performed by utilizing a compound that includes a peptide orpeptidomimetic, e.g., containing the amino acid sequence[L/F/I]XR[Q/S/T]L[S/T][Hydrophobic] (SEQ ID NO: 17), wherein Xrepresents any amino acid and the peptide or peptidomimetic includes nomore than 50 amino acids. Hydrophobic amino acids are selected from thegroup consisting of alanine, cysteine, isoleucine, leucine, methionine,phenylalanine, proline, tryptophan, tyrosine, and valine. In oneembodiment, the peptide or peptidomimetic includes the amino acidsequence LQRQLSI (SEQ ID NO: 16).

In one aspect of the invention, administering a compound that is capableof inhibiting an activity of a MAPKAP kinase-2 polypeptide for treatinga cellular proliferative disorder in a patient sensitizes p53-deficientcells to chemotherapeutic challenge. This can result in increasedlikelihood of death of aberrantly proliferating cells in a patient, incomparison to p53-deficient cells in a patient not receiving a MAPKAPkinase-2 inhibitor. The chemotherapeutics can be administeredsubsequently or concomitantly to administration of a MAPKAP kinase-2inhibitor. An exemplary chemotherapeutic used in this regimen is UCN-01.Increased chemosensitivity of p53-deficient cells to a MAPKAP kinase-2treatment may alter a DNA damage-responsive cell cycle checkpoint, incomparison to control p-53 deficient cells in a control patient notreceiving the described therapy. Alteration of a DNA damage-responsivecell cycle checkpoint may occur at G₁/S phase arrest, where Cdc25adegradation impaired. Alteration of a DNA damage-responsive cell cyclecheckpoint may occur at G₂/M phase arrest, where, the interactionbetween Cdc25b and a 14-3-3 protein is reduced. The above-mentionedalterations of cell cycle checkpoints may increase the likelihood ofcell death, including cell death by apoptosis.

The invention further features kits that include: (a) a means ofdetecting the level of p53 polypeptide expression or activity in acellular sample, e.g., by using an anti-p53 antibody; and (b) a compoundthat is capable of inhibiting an activity of a MAPKAP kinase-2polypeptide, e.g., UCN-01. In some instances, the kit can also containone or more chemotherapeutic agents.

The invention additionally features a method for treating a cellularproliferative disorder in a patient including administering to thepatient a compound that is capable of inhibiting an activity of a MAPKAPkinase-2 polypeptide. In some instances, the cellular proliferativedisorder includes the presence of one or more p53-deficient cells, e.g.,tumor cells, in the patient. Administration of a MAPKAP kinase-2polypeptide together with a chemotherapeutic compound has the desiredeffect of reducing tumor size. Administration of the described therapycan be, e.g., by direct injection into the tumor.

The invention further features a method for identifying a compound thatmay be an inhibitor of substrate binding to a MAPKAP kinase-2polypeptide, the method including the steps of: contacting the MAPKAPkinase-2 polypeptide and a compound capable of binding the MAPKAPkinase-2 polypeptide under conditions allowing the formation of acomplex between the compound and the MAPKAP kinase-2 polypeptide;contacting the complex with a candidate compound; and measuring thedisplacement of the compound capable of binding the MAPKAP kinase-2polypeptide from the MAPKAP kinase-2 polypeptide. The displacement ofthe compound capable of binding identifies the candidate compound as acompound that may be an inhibitor of substrate binding to a MAPKAPkinase-2 polypeptide. In one embodiment, the compound capable of bindingthe MAPKAP kinase-2 polypeptide includes a peptide or peptidomimetic,e.g., containing the amino acid sequence[L/F/I]XR[Q/S/T]L[S/T][Hydrophobic] (SEQ ID NO: 17), wherein the peptideor peptidomimetic includes no more than 50 amino acids. For example, thepeptide or peptidomimetic may include the amino acid sequence LQRQLSI(SEQ ID NO: 16). In the foregoing method, a substrate-binding fragmentof a MAPKAP kinase-2 polypeptide may be utilized in place of afull-length MAPKAP kinase-2 polypeptide.

Variations of the foregoing aspect are also possible in the methods ofthe invention. The MAPKAP kinase-2 polypeptide, or substrate-bindingfragment thereof, and compound capable of binding the polypeptide may becontacted in the presence of a candidate compound, and any means ofmeasuring the binding of the MAPKAP kinase-2 polypeptide and thecompound capable of binding may be used in the methods of the invention.In general, if the amount of binding of the MAPKAP kinase-2 polypeptideand the compound capable of binding is decreased in the presence of thecandidate compound in comparison to the amount of binding measured inthe absence of the candidate compound, then the candidate compound isdetermined to be an inhibitor of substrate binding using the methods ofthe invention.

In another aspect, the invention features a method for identifying acompound that may be an inhibitor of substrate binding to a MAPKAPkinase-2 polypeptide or substrate-binding fragment thereof, the methodincluding the steps of: providing a three-dimensional model of theMAPKAP kinase-2 polypeptide having at least one atomic coordinate, orsurrogate thereof, from Table 1 for at least three of the residuesIle74, Glu145, Lys188, Glu190, Phe210, Cys224, Tyr225, Thr226, Pro227,Tyr228, Tyr229, and Asp345, or atomic coordinates that have a root meansquare deviation of the coordinates of less than 3 Å; and producing astructure for a candidate compound, the structure defining a moleculehaving sufficient surface complementary to the MAPKAP kinase-2polypeptide to bind the MAPKAP kinase-2 polypeptide in an aqueoussolution.

The invention further features a compound that includes a peptide orpeptidomimetic, e.g., containing the amino acid sequence[L/F/I]XR[Q/S/T]L[S/T][Hydrophobic] (SEQ ID NO: 17), wherein the peptideor peptidomimetic includes no more than 50 amino acids. In oneembodiment, the peptide or peptidomimetic includes the amino acidsequence LQRQLSI (SEQ ID NO: 16). An inhibitory compound of theinvention may include a covalently-linked moiety capable oftranslocating across a biological membrane, such as a penetratin or TATpeptide. Alternatively, such a compound may be administered in the formof a prodrug.

In another aspect, the invention features a pharmaceutical compositionfor treating a cellular proliferative disorder in a patient, thecomposition including: a compound that is capable of inhibiting anactivity of a MAPKAP kinase-2 polypeptide; and a chemotherapeutic agent,wherein the composition is formulated in an amount sufficient to treatthe cellular proliferative disorder. Any chemotherapeutic agent known inthe art may be useful in the compositions of the invention. Aninhibitory compound useful in the pharmaceutical composition may includea covalently-linked moiety capable of translocating across a biologicalmembrane, such as a penetratin or TAT peptide. Alternatively, such acompound may be administered in the form of a prodrug. Any compoundsdescribed in any of the foregoing aspects, including small moleculeinhibitors, compounds containing siNA molecules, antisense RNAmolecules, or peptides, may be useful in the pharmaceutical compositionsof the invention.

In any of the foregoing aspects of the invention, it is desirable thatthe inhibitory compounds be specific inhibitors of MAPKAP kinase-2,e.g., compounds that inhibit a MAPKAP kinase-2 polypeptide without alsosubstantially inhibiting related kinases such as Chk1, Chk2, and p38SAPK, although compounds that inhibit a MAPKAP kinase-2 polypeptide in aless selective or non-selective manner are also useful in the methods ofthe invention.

As used throughout this specification and the appended claims, thefollowing terms have the meanings specified.

By an “amino acid fragment” is meant an amino acid residue that has beenincorporated into a peptide chain via its alpha carboxyl, its alphanitrogen, or both. A terminal amino acid is any natural or unnaturalamino acid residue at the amino-terminus or the carboxy-terminus. Aninternal amino acid is any natural or unnatural amino acid residue thatis not a terminal amino acid.

By “analog” is meant a molecule that is not identical but has analogousfeatures. For example, a polypeptide analog retains the biologicalactivity of a corresponding naturally-occurring polypeptide, whilehaving certain biochemical modifications that enhance the analog'sfunction relative to a naturally occurring polypeptide. Such biochemicalmodifications could increase the analog's protease resistance, membranepermeability, or half-life, without altering, for example, ligandbinding. An analog may include an unnatural amino acid.

By “antisense,” as used herein in reference to nucleic acids, is meant anucleic acid sequence, regardless of length, that is complementary tothe coding strand of a gene.

By “atomic coordinates” is meant those three-dimensional coordinates ofthe atoms in a crystalline material derived from mathematical equationsrelated to the patterns obtained on diffraction of x-rays by the atoms(x-ray scattering centers) of the crystalline material. The diffractiondata are used to calculate an electron density map of the unit cell ofthe crystal. These electron density maps are used to establish thepositions of the individual atoms within the unit cell of the crystal.Atomic coordinates can be transformed, as is known to those skilled inthe art, to different coordinate systems (i.e., surrogate systems)without affecting the relative positions of the atoms.

By “binding to” a molecule is meant having a physicochemical affinityfor that molecule. Binding may be measured by any of the methods of theinvention, e.g., using an in vitro translation binding assay.

By “biological activity” of a polypeptide or other compound is meanthaving structural, regulatory, or biochemical functions of a naturallyoccurring molecule. For example, one biological activity of a MAPKAPkinase-2 polypeptide is substrate binding, e.g., peptide binding, whichmay be measured using in vivo or in vitro binding assays.

By “caged compound” is meant a biologically active molecule coupled to acleavable moiety such that the resulting coupled compound lacksbiological activity as long as the moiety remains attached. Such amoiety prevents bioaction by sterically shielding one or more chemicalgroups of the molecule. The moiety may be removed by any means,including enzymatic, chemical, or photolytic; removal of the moietyresults in restoration of the molecule's biological activity.

By “candidate compound” is meant any nucleic acid molecule, polypeptide,or other small molecule that is assayed for its ability to alter gene orprotein expression levels, or the biological activity of a gene orprotein by employing one of the assay methods described herein.Candidate compounds include, for example, peptides, polypeptides,synthesized organic molecules, naturally occurring organic molecules,nucleic acid molecules, and components thereof.

By “cellular proliferative disorder” is meant any pathological conditionin which there is an abnormal increase or decrease in cellproliferation. Exemplary cellular proliferative disorders include canceror neoplasms, inflammatory diseases, or hyperplasias (e.g., some formsof hypertension, prostatic hyperplasia).

By “chemotherapeutic agent” is meant one or more chemical agents used inthe treatment or control of proliferative diseases, including cancer.Chemotherapeutic agents include cytotoxic and cytostatic agents.

By “complex” is meant a chemical association of two or more molecules.Complexes may include a network of weak electrostatic bonds thatmaintain the association of the molecules. Other types of interactions,such as covalent, ionic, hydrogen bond, hydrophobic, or van der Waalsinteractions, may be present instead of or in addition to electrostaticbonds between members of a complex.

By “computer modeling” is meant the application of a computationalprogram to determine one or more of the following: the location andbinding proximity of a ligand to a binding moiety, the occupied space ofa bound ligand, the amount of complementary contact surface between abinding moiety and a ligand, the deformation energy of binding of agiven ligand to a binding moiety, and some estimate of hydrogen bondingstrength, van der Waals interaction, hydrophobic interaction, and/orelectrostatic interaction energies between ligand and binding moiety.Computer modeling can also provide comparisons between the features of amodel system and a candidate compound. For example, a computer modelingexperiment can compare a pharmacophore model of the invention with acandidate compound to assess the fit of the candidate compound with themodel. Examples of techniques useful in the above evaluations include:quantum mechanics, molecular mechanics, molecular dynamics, Monte Carlosampling, systematic searches and distance geometry methods. Furtherdescriptions of computer modeling programs are provided elsewhereherein.

By “detectably-labeled” is meant any means for marking and identifyingthe presence of a molecule, e.g., a peptide or a peptidomimetic smallmolecule that interacts with a MAPKAP kinase-2 domain. Methods fordetectably-labeling a molecule are well known in the art and include,without limitation, radionuclides (e.g., with an isotope such as ³²P,³³P, ¹²⁵I, or ³⁵S), nonradioactive labeling (e.g., chemiluminescentlabeling or fluorescein labeling), and epitope tags.

If required, molecules can be differentially labeled using markers thatcan distinguish the presence of multiply distinct molecules. Forexample, a peptide that interacts with a MAPKAP kinase-2 domain can belabeled with fluorescein and a MAPKAP kinase-2 domain can be labeledwith Texas Red. The presence of the peptide can be monitoredsimultaneously with the presence of the MAPKAP kinase-2 domain.

By “fragment” is meant a portion of a polypeptide or nucleic acid havinga region that is substantially identical to a portion of a referenceprotein or nucleic acid and retains at least 50%, 75%, 80%, 90%, 95%, oreven 99% of at least one biological activity of the reference protein ornucleic acid.

By “hydrophobic” in the context of amino acids is meant any of thefollowing amino acids: alanine, cysteine, isoleucine, leucine,methionine, phenylalanine, proline, tryptophan, tyrosine, or valine.

By “inhibit an activity of a MAPKAP kinase-2 polypeptide” is meant toreduce one or more biological activities of MAPKAP kinase-2 polypeptide.Desirably, the inhibition is a decrease of at least 10%, 20%, 30%, 40%,50%, 60%, 70%, 80%, 90% or 95% in biological activity, relative to acontrol activity, for example the expression or substrate-bindingcapability of a naturally occurring MAPKAP kinase-2 polypeptide. Anexample of a compound that inhibits a MAPKAP kinase-2 polypeptide isUCN-01.

By “MAPKAP kinase-2 biological activity” is meant any activity known tobe caused in vivo or in vitro by a MAPKAP kinase-2 polypeptide. Forexample, such activity could be caused by at least one of the following:function in a DNA damage response pathway, cell cycle control,transcriptional regulation, chromatin remodeling, or substrate binding.In one assay for MAPKAP kinase-2 biological activity, the ability ofMAPKAP kinase-2, or a fragment or mutant thereof comprising asubstrate-binding domain, to bind a substrate is measured.

By “MAPKAP kinase-2 nucleic acid” is meant a nucleic acid that encodesall or a portion of a MAPKAP kinase-2 polypeptide or is substantiallyidentical to all or a portion of the nucleic acid sequence of GenbankAccession Nos. NM_(—)004759 (SEQ ID NO: 1) or NM_(—)032960 (SEQ ID NO:2), or analog thereof.

By “MAPKAP kinase-2 polypeptide” and “MK2” are used interchangeablyherein, and denote a polypeptide substantially identical to all or aportion of the polypeptide sequence of Genbank Accession Nos.NP_(—)004750 (SEQ ID NO: 3) or P49137 (SEQ ID NO: 4), or analog thereof,and having MAPKAP kinase-2 biological activity.

By “neoplasia” or “neoplasm” is meant a disease characterized by thepathological proliferation of a cell or tissue and its subsequentmigration to or invasion of other tissues or organs. Neoplasia growth istypically uncontrolled and progressive, and occurs under conditions thatwould not elicit, or would cause cessation of, multiplication of normalcells. Neoplasias can affect a variety of cell types, tissues, ororgans, including but not limited to an organ selected from the groupconsisting of bladder, bone, brain, breast, cartilage, glia, esophagus,fallopian tube, gallbladder, heart, intestines, kidney, liver, lung,lymph node, nervous tissue, ovaries, pancreas, prostate, skeletalmuscle, skin, spinal cord, spleen, stomach, testes, thymus, thyroid,trachea, urogenital tract, ureter, urethra, uterus, and vagina, or atissue or cell type thereof. Neoplasias include cancers, such asacoustic neuroma, acute leukemia, acute lymphocytic leukemia, acutemonocytic leukemia, acute myeloblastic leukemia, acute myelocyticleukemia, acute myelomonocytic leukemia, acute promyelocytic leukemia,acute erythroleukemia, adenocarcinoma, angiosarcoma, astrocytoma, basalcell carcinoma, bile duct carcinoma, bladder carcinoma, brain cancer,breast cancer, bronchogenic carcinoma, cervical cancer, chondrosarcoma,chordoma, choriocarcinoma, chronic leukemia, chronic lymphocyticleukemia, chronic myelocytic leukemia, colon cancer, colon carcinoma,craniopharyngioma, cystadenocarcinoma, embryonal carcinoma,endotheliosarcoma, ependymoma, epithelial carcinoma, Ewing's tumor,glioma, heavy chain disease, hemangioblastoma, hepatoma, Hodgkin'sdisease, large cell carcinoma, leiomyosarcoma, liposarcoma, lung cancer,lung carcinoma, lymphangioendotheliosarcoma, lymphangiosarcoma,macroglobulinemia, medullary carcinoma, medulloblastoma, melanoma,meningioma, mesothelioma, myxosarcoma, neuroblastoma, non-Hodgkin'sdisease, oligodendroglioma, osteogenic sarcoma, ovarian cancer,pancreatic cancer, papillary adenocarcinomas, papillary carcinoma,pinealoma, polycythemia vera, prostate cancer, rhabdomyosarcoma, renalcell carcinoma, retinoblastoma, schwannoma, sebaceous gland carcinoma,seminoma, small cell lung carcinoma, squamous cell carcinoma, sweatgland carcinoma, synovioma, testicular cancer, uterine cancer,Waldenstrom's fibrosarcoma, and Wilm's tumor.

By “nucleic acid” is meant an oligomer or polymer of ribonucleic acid ordeoxyribonucleic acid, or analog thereof. This term includes oligomersconsisting of naturally occurring bases, sugars, and intersugar(backbone) linkages as well as oligomers having non-naturally occurringportions which function similarly. Such modified or substitutedoligonucleotides are often preferred over native forms because ofproperties such as, for example, enhanced cellular uptake and increasedstability in the presence of nucleases.

Specific examples of some preferred nucleic acids may containphosphorothioates, phosphotriesters, methyl phosphonates, short chainalkyl or cycloalkyl intersugar linkages or short chain heteroatomic orheterocyclic intersugar linkages. Most preferred are those withCH₂—NH_CH₂, CH₂—N(CH₃)—O—CH₂, CH₂—O—N(CH₃)—CH₂, CH₂—N(CH₃)—N(CH₃)—CH₂and O—N(CH₃)—CH₂—CH₂ backbones (where phosphodiester is O—P—CH₂). Alsopreferred are oligonucleotides having morpholino backbone structures(Summerton, J. E. and Weller, D. D., U.S. Pat. No. 5,034,506). In otherpreferred embodiments, such as the protein-nucleic acid (PNA) backbone,the phosphodiester backbone of the oligonucleotide may be replaced witha polyamide backbone, the bases being bound directly or indirectly tothe aza nitrogen atoms of the polyamide backbone (P. E. Nielsen et al.Science 199: 254, 1997). Other preferred oligonucleotides may containalkyl and halogen-substituted sugar moieties comprising one of thefollowing at the 2′ position: OH, SH, SCH₃, F, OCN, O(CH₂)_(n)NH₂ orO(CH₂)_(n) CH₃, where n is from 1 to about 10; C₁ to C₁₀ lower alkyl,substituted lower alkyl, alkaryl or aralkyl; Cl; Br; CN; CF₃; OCF₃; O-,S-, or N-alkyl; O-, S-, or N-alkenyl; SOCH₃; SO₂CH₃; ONO₂; NO₂; N₃; NH₂;heterocycloalkyl; heterocycloalkaryl; aminoalkylamino; polyalkylamino;substituted silyl; an RNA cleaving group; a conjugate; a reporter group;an intercalator; a group for improving the pharmacokinetic properties ofan oligonucleotide; or a group for improving the pharmacodynamicproperties of an oligonucleotide and other substituents having similarproperties. Oligonucleotides may also have sugar mimetics such ascyclobutyls in place of the pentofuranosyl group.

Other preferred embodiments may include at least one modified base form.Some specific examples of such modified bases include 2-(amino)adenine,2-(methylamino)adenine, 2-(imidazolylalkyl)adenine,2-(aminoalklyamino)adenine, or other heterosubstituted alkyladenines.

By “p53-deficient cell” is meant a cell expressing substantially lessp53 polypeptide, or exhibiting substantially less p53 polypeptideactivity, e.g., 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 98%,99%, or even 100% less p53 expression or activity, than a correspondingwild-type cell. For example, p53-deficient cells, e.g., tumor cells, arepresent in some neoplastic disorders. p53-deficient cells include cellswith one or more p53 gene mutations, e.g., point mutations or nullmutations, that reduce or eliminate expression or activity.

By a “peptidomimetic” is meant a compound that is capable of mimickingor antagonizing the biological actions of a natural parent peptide. Apeptidomimetic may include non-peptidic structural elements, unnaturalpeptides, synthesized organic molecules, naturally occurring organicmolecules, nucleic acid molecules, and components thereof.Identification of a peptidomimetic can be accomplished by screeningmethods incorporating a binding pair and identifying compounds thatdisplace the binding pair. Alternatively, a peptidomimetic can bedesigned in silico, by molecular modeling of a known protein-proteininteraction, for example, the interaction of a peptide of the inventionand a MAPKAP kinase-2 domain. In one embodiment, the peptidomimetic willdisplace one member of a binding pair by occupying the same bindinginterface. It is desirable that the peptidomimetic have a higher bindingaffinity to the binding interface.

By “pharmaceutically acceptable excipient” is meant a carrier that isphysiologically acceptable to the subject to which it is administeredand that preserves the therapeutic properties of the compound with whichit is administered. One exemplary pharmaceutically acceptable excipientis physiological saline. Other physiologically acceptable excipients andtheir formulations are known to one skilled in the art and described,for example, in “Remington: The Science and Practice of Pharmacy,” (20thed., ed. A. R. Gennaro, 2000, Lippincott Williams & Wilkins).

By “phosphopeptide” is meant a peptide in which one or more phosphatemoieties are covalently linked to serine, threonine, tyrosine, asparticacid, histidine amino acid residues, or amino acid analogs. A peptidecan be phosphorylated to the extent of the number of serine, threonine,tyrosine, or histidine amino acid residues that is present. Aphosphopeptide may be phosphorylated at four independent Ser/Thr/Tyrresidues, at three independent Ser/Thr/Tyr residues, or at twoindependent Ser/Thr/Tyr residues. Desirably, a phosphopeptide isphosphorylated at one Ser/Thr/Tyr residue regardless of the presence ofmultiple Ser, Thr, or Tyr residues.

Typically, a phosphopeptide is produced by expression in a prokaryoticor eukaryotic cell under appropriate conditions or in translationextracts where the peptide is subsequently isolated, and phosphorylatedusing an appropriate kinase. Alternatively, a phosphopeptide may besynthesized by standard chemical methods, for example, usingN-α-FMOC-protected amino acids (including appropriate phosphoaminoacids). In a desired embodiment, the use of non-hydrolysable phosphateanalogs can be incorporated to produce non-hydrolysable phosphopeptides(Jenkins et al., J. Am. Chem. Soc., 124:6584-6593, 2002; hereinincorporated by reference). Such methods of protein synthesis arecommonly used and practiced by standard methods in molecular biology andprotein biochemistry (Ausubel et al., Current Protocols in MolecularBiology, John Wiley & Sons, New York, N.Y., 1994, J. Sambrook and D.Russel, Molecular Cloning: A Laboratory Manual, 3^(rd) Edition, ColdSpring Harbor Laboratory Press, Woodbury N.Y., 2000). In one embodiment,a phosphopeptide is generally not longer than 100 amino acid residues inlength. Shorter phosphopeptides, e.g., less than 50, 25, 20, or 15residues, are also possible. Phosphopeptides may be as short as 2, 3, 4,5, 6, 7, 8, 9, or 10 amino acid residues long.

By “protein” or “polypeptide” or “peptide” is meant any chain of morethan two natural or unnatural amino acids, regardless ofpost-translational modification (e.g., glycosylation orphosphorylation), constituting all or part of a naturally-occurring ornon-naturally occurring polypeptide or peptide, as is described herein.As used herein, a natural amino acid is a natural α-amino acid havingthe L-configuration, such as those normally occurring in naturalproteins. Unnatural amino acid refers to an amino acid, which normallydoes not occur in proteins, e.g., an epimer of a natural α-amino acidhaving the L configuration, that is to say an amino acid having theunnatural D-configuration; or a (D,L)-isomeric mixture thereof; or ahomologue of such an amino acid, for example, a β-amino acid, anα,α-disubstituted amino acid, or an α-amino acid wherein the amino acidside chain has been shortened by one or two methylene groups orlengthened to up to 10 carbon atoms, such as an α-amino alkanoic acidwith 5 up to and including 10 carbon atoms in a linear chain, anunsubstituted or substituted aromatic (α-aryl or α-aryl lower alkyl),for example, a substituted phenylalanine or phenylglycine. Other aminoacids that may also be incorporated into a polypeptide include ornithine(O or Orn) and hydroxyproline (Hyp).

Polypeptides or derivatives thereof may be fused or attached to anotherprotein or peptide, for example, as a Glutathione-S-Transferase (GST)fusion polypeptide. Other commonly employed fusion polypeptides include,but are not limited to, maltose-binding protein, Staphylococcus aureusprotein A, Flag-Tag, HA-tag, green fluorescent proteins (e.g., eGFP,eYFP, eCFP, GFP, YFP, CFP), red fluorescent protein, polyhistidine(6×His), and cellulose-binding protein.

By “prodrug” is meant a compound that is modified in vivo, resulting information of a biologically active drug compound, for example byhydrolysis in blood. A thorough discussion of prodrug modifications isprovided in T. Higuchi and V. Stella, Pro-drugs as Novel DeliverySystems, Vol. 14 of the A.C.S. Symposium Series, Edward B. Roche, ed.,Bioreversible Carriers in Drug Design, American PharmaceuticalAssociation and Pergamon Press, 1987, and Judkins et al., SyntheticCommunications 26(23):4351-4367, 1996, each of which is incorporatedherein by reference.

By “purified” is meant separated from other components that naturallyaccompany it. Typically, a factor is substantially pure when it is atleast 50%, by weight, free from proteins, antibodies, andnaturally-occurring organic molecules with which it is naturallyassociated. The factor may be at least 75%, 90%, or even 99%, by weight,pure. A substantially pure factor may be obtained by chemical synthesis,separation of the factor from natural sources, or production of thefactor in a recombinant host cell that does not naturally produce thefactor. Proteins, vesicles, and organelles may be purified by oneskilled in the art using standard techniques such as those described byColigan et al. (Current Protocols in Protein Science, John Wiley & Sons,New York, 2000). The factor is desirably at least 2, 5, or 10 times aspure as the starting material, as measured using polyacrylamide gelelectrophoresis or column chromatography (including HPLC) analysis(Coligan et al., supra). Exemplary methods of purification include (i)salting-out, i.e., (NH₄)₂SO₄ precipitation; (ii) conventionalchromatography, e.g., ion exchange, size exclusion, hydrophobicinteraction, or reverse-phase; (iii) affinity chromatography, e.g.,immunoaffinity, active site affinity, dye affinity, or immobilized-metalaffinity; and (iv) preparative electrophoresis, e.g., isoelectricfocusing or native PAGE.

By “RNA interference” (RNAi) is meant a phenomenon where double-strandedRNA homologous to a target mRNA leads to degradation of the targetedmRNA. More broadly defined as degradation of target mRNAs by homologoussiRNAs.

By “sensitivity” or “sensitivity to an agent” is meant an increasedlikelihood of cell death in response to genotoxic stress. An exemplarymeans of sensitivity occurs when a patient having p53-deficient tumorcell is administered a composition including MAPKAP kinase-2 polypeptideinhibitor and a chemotherapeutic agent, resulting in reduction of tumorsize. A reduction in tumor size in the described patient receiving thedescribed therapy is determined in comparison to a control p53-deficienttumor cell in a control patient not receiving the described therapy.Desirably, tumors are reduced is size by 10%, 20%, 30%, 40%, 50%, 60%,70%, 80%, 90% or 95% in comparison to the described control.

By “siNA” is meant small interfering nucleic acids. One exemplary siNAis composed of ribonucleic acid (siRNA). siRNAs can be 21-25 nt RNAsderived from processing of linear double-stranded RNA. siRNAs assemblein complexes termed RISC(RNA-induced silencing complex) and targethomologous RNA sequences for endonucleolytic cleavage. Synthetic siRNAsalso recruit RISCs and are capable of cleaving homologous RNA sequences

By “specifically inhibit an activity of a MAPKAP kinase-2 polypeptide”is meant to reduce one or more biological activities of MAPKAP kinase-2polypeptide, without substantially inhibiting related kinases, e.g.,Chk1, Chk2, and p38 SAPK. Desirably, the specific inhibition is adecrease of at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% or 95%in biological activity, relative to a control activity, for example theexpression or substrate-binding capability of a naturally occurringMAPKAP kinase-2 polypeptide. An exemplary means of specific inhibitionoccurs through use of RNA interference. An example of a compound thatinhibits a MAPKAP kinase-2 polypeptide, but does not do so specifically,is UCN-01.

By “substantially identical” is meant a polypeptide or nucleic acidexhibiting at least 75%, 85%, 90%, 95%, or even 99% identity to areference amino acid or nucleic acid sequence. For polypeptides, thelength of comparison sequences will generally be at least 35 aminoacids, 45 amino acids, 55 amino acids, or even 70 amino acids. Fornucleic acids, the length of comparison sequences will generally be atleast 60 nucleotides, 90 nucleotides, or even 120 nucleotides.

Sequence identity is typically measured using publicly availablecomputer programs. Computer program methods to determine identitybetween two sequences include, but are not limited to, the GCG programpackage (Devereux et al., Nucleic Acids Research 12: 387, 1984), BLASTP,BLASTN, and FASTA (Altschul et al., J. Mol. Biol. 215:403 (1990). Thewell-known Smith Waterman algorithm may also be used to determineidentity. The BLAST program is publicly available from NCBI and othersources (e.g., BLAST Manual, Altschul et al., NCBI NLM NIH, Bethesda,Md. 20894). These software programs match similar sequences by assigningdegrees of homology to various substitutions, deletions, and othermodifications. Conservative substitutions for amino acid comparisonstypically include substitutions within the following groups: glycine,alanine, valine, isoleucine, leucine; aspartic acid, glutamic acid,asparagine, glutamine; serine, threonine; lysine, arginine; andphenylalanine, tyrosine.

By “substantially inhibit” is meant to reduce one or more activities ofthe molecule being inhibited by at least 50%, 60%, 70%, 80%, 90%, 95%,or even 98% compared to a control activity value.

By “substrate-binding fragment” in reference to a MAPKAP kinase-2polypeptide is meant a portion of the polypeptide that is capable ofbinding a peptide or peptidomimetic substrate. For example, fragments ofMAPKAP kinase-2 polypeptide that include the region Phe46-Asp345 (withreference to SEQ ID NO: 3) are substrate-binding fragments.

By “surrogate,” in the context of atomic coordinates, is meant anymodification (e.g., mathematical modification or scaling) of thecoordinates that preserves the relative relationships among thecoordinates.

By “three-dimensional model” is meant a three-dimensional representationof a molecule's structure. Computer modeling may be used to generatesuch a model in conjunction with structural data. These data couldinclude x-ray crystallographic data, nuclear magnetic resonance data,electron microscopy data, or any other source of experimental ortheoretical data useful for generating a model of a molecule or complexof molecules.

By “treating” a disease, disorder, or condition is meant preventing ordelaying an initial or subsequent occurrence of a disease, disorder, orcondition; increasing the disease-free survival time between thedisappearance of a condition and its reoccurrence; stabilizing orreducing an adverse symptom associated with a condition; or inhibiting,slowing, or stabilizing the progression of a condition. Desirably, atleast 20, 40, 60, 80, 90, or 95% of the treated subjects have a completeremission in which all evidence of the disease disappears. In anotherdesirable embodiment, the length of time a patient survives after beingdiagnosed with a condition and treated with a compound of the inventionis at least 20, 40, 60, 80, 100, 200, or even 500% greater than (i) theaverage amount of time an untreated patient survives or (ii) the averageamount of time a patient treated with another therapy survives.

By “unnatural amino acid” is meant an organic compound that has astructure similar to a natural amino acid, where it mimics the structureand reactivity of a natural amino acid. The unnatural amino acid asdefined herein generally increases or enhances the properties of apeptide (e.g., selectivity, stability, binding affinity) when theunnatural amino acid is either substituted for a natural amino acid orincorporated into a peptide. Unnatural amino acids and peptidesincluding such amino acids are described, e.g., in U.S. Pat. Nos.6,566,330 and 6,555,522.

Other features and advantages of the invention will be apparent from thefollowing description of the desirable embodiments thereof, and from theclaims.

BRIEF DESCRIPTION OF THE DRAWINGS

FIGS. 1A-1E depict the substrate specificity and kinetic analysis ofsubstrate phosphorylation by p38α SAPK. FIG. 1A is a table showing p38substrate specificity determined using oriented peptide libraryscreening. Residues displaying the highest selectivity are shown; thosewith selection values >1.7 in bold. Abbreviations: MEF2A, MyocyteEnhancer Factor 2; ATF2, Activating Transcription Factor 2; 3PK1, MAPKinase-activated Protein Kinase-3. FIG. 1B is a graph showing thekinetics of in vitro phosphorylation of an optimal p38 peptide (p38tide)and a peptide from p47phox (p47tide) by p38α kinase. FIG. 1C is a graphshowing the kinetics of in vitro phosphorylation of wild-typeGST-p47phox, the Ser345→Ala mutant, and the Ser348→Ala mutant. Typicaldata from n=3 experiments is shown. FIG. 1D shows the in vitrophosphorylation of full-length wild-type or mutant p47phox proteins.Samples were analyzed by SDS-PAGE/autoradiography. FIG. 1E is a table ofkinetic parameters for the reactions shown in FIG. 1C.

FIGS. 2A-2C depict the involvement of MAPKAP kinase-2 in thephosphorylation of Cdc25B and Cdc25C after DNA damage. FIG. 2A shows thephosphorylation of full-length wild type Cdc25B or a Ser−323→Ala mutantby p38α SAPK or MAPKAP kinase-2. After phosphorylation, generation of a14-3-3 binding site on Cdc25B was determined by a 14-3-3-MBP pulldownfollowed by SDSPAGE/autoradiography. In FIG. 2B, the kinetics of MAPKAPkinase-2 phosphorylation and generation of a 14-3-3-binding site onCdc25B were measured in U2OS cells following 20 J/m² UV-irradiation. InFIG. 2C, signaling events in the G₂/M, G₁, and S-phase checkpointresponse were analyzed in GFP siRNA- or MAPKAP kinase-2 siRNA-treatedU2OS cells before and two hours after UV-induced DNA damage. Equalloading was determined by western blotting for β-actin.

FIGS. 3A-3C depict the substrate specificity and kinetic analysis ofsubstrate phosphorylation by MAPKAP kinase-2. FIG. 3A is a table showingMAPKAP kinase-2 substrate specificity determined by oriented peptidelibrary screening. Abbreviations: HSP27, Heat Shock protein 27; 5-LO,5-Lipoxygenase; LSP1, lymphocyte-specific protein; SRF, Serum ResponseFactor; GS, Glycogen Synthase; TH, Tyrosine Hydroxylase. FIG. 3B is agraph showing the kinetics of in vitro phosphorylation of the optimalMAPKAP kinase-2 peptide (MK2tide) by MAPKAP kinase-2. FIG. 3C is a tableof kinetic parameters for MAPKAP kinase-2 phosphorylation of wild-typeand mutant MK2tides.

FIGS. 4A-4K show that MAPKAP kinase-2 is required for G₂/M arrestfollowing DNA damage. FIG. 4A shows that UV-C irradiation induces DNAdamage as revealed by nuclear foci formation. U2OS cells were mockirradiated or exposed to 20 J/m² of UV-C radiation and immunostained twohours later using an anti-phospho(Ser/Thr) ATM/ATR substrate antibody.FIG. 4B is a graph depicting a FACS analysis of GFP siRNA-treatednon-irradiated U2OS cells placed in 50 ng/ml nocodazole-containing mediafor 16 hours. Cells were analyzed for DNA content by PI staining. FIG.4C is a graph depicting a FACS analysis of GFP siRNA-treatednon-irradiated U2OS cells placed in 50 ng/ml nocodazole-containing mediafor 16 hours. Cells were analyzed for phospho-histone H3 staining as amarker of mitotic entry. FIG. 4D is a graph depicting a FACS analysis ofGFP siRNA-treated U2OS cells irradiated as described for FIG. 4A andthen placed in 50 ng/ml nocodazole-containing media for an additional 16hours. Cells were analyzed for DNA content by PI staining. FIG. 4E is agraph depicting a FACS analysis of GFP siRNA-treated U2OS cellsirradiated as described for FIG. 4A and then placed in 50 ng/mlnocodazole-containing media for an additional 16 hours. Cells wereanalyzed for phospho-histone H3 staining as a marker of mitotic entry.FIG. 4F is a graph depicting a FACS analysis of MAPKAP kinase-2siRNA-treated non-irradiated U2OS cells placed in 50 ng/mlnocodazole-containing media for 16 hours. Cells were analyzed for DNAcontent by PI staining. FIG. 4G is a graph depicting a FACS analysis ofMAPKAP kinase-2 siRNA-treated non-irradiated U2OS cells placed in 50ng/ml nocodazole-containing media for 16 hours. Cells were analyzed forphospho-histone H3 staining as a marker of mitotic entry. FIG. 4H is agraph depicting a FACS analysis of MAPKAP kinase-2 siRNA-treated U2OScells irradiated as described for FIG. 4A and then placed in 50 ng/mlnocodazole-containing media for 16 hours. Cells were analyzed for DNAcontent by PI staining. FIG. 4I is a graph depicting a FACS analysis ofMAPKAP kinase-2 siRNA-treated U2OS cells irradiated as described forFIG. 4A and then placed in 50 ng/ml nocodazole-containing media for 16hours. Cells were analyzed for phospho-histone H3 staining as a markerof mitotic entry. FIG. 4J is a graph depicting the results of anexperiment in which GFP siRNA- or MAPKAP kinase-2-siRNA treated U2OScells were either mock treated or exposed to 20 J/m² of UV-Cirradiation, and analyzed as described for FIGS. 4B-4I. Representativeresults of each experiment are shown. FIG. 4K is a graph depicting theresults of an experiment in which GFP siRNA- or MAPKAP kinase-2-siRNAtreated U2OS cells were either mock treated or exposed to 10 Gy ofionizing radiation, and analyzed as described for FIGS. 4B-4I.Representative results of each experiment are shown.

FIGS. 5A-5E show that MAPKAP kinase-2 is required for S-phase arrest andcell survival following DNA damage. In FIG. 5A, GFP siRNA- or MAPKAPkinase-2-siRNA-treated U2OS cells were mock treated or UV-irradiated andallowed to recover for 30 min. BrdU was added and cells were fixed andanalyzed by FACS for DNA content and BrdU incorporation twelve hourslater. FIG. 5B is a graph showing the percentage of cells in FIG. 5Ashowing BrdU incorporation at two and twelve hours following BrdUaddition. In FIG. 5C, GFP siRNA- or MAPKAP kinase-2-siRNA-treated U2OScells were either mock treated or UV-irradiated, allowed to recover for30 min, then pulse-labeled with BrdU for 30 minutes. At the indicatedtimes after irradiation the distribution of DNA content was analyzed inthe BrdU-positive population. In FIG. 5D, GFP siRNA- or MAPKAPkinase-2-siRNA treated U2OS cells were either mock treated or irradiatedat the indicated UV-C dose. Cells were stained with Crystal Violetforty-eight hours later and visualized. Insets show a magnified view.FIG. 5E is a graph showing the results of quantitative colony formingassays performed by plating cells at a density of ˜100 cells per 35 mm²dish. Cells were treated as in FIG. 5D, and assays were performed intriplicate for each condition.

FIGS. 6A-6E show a comparison of active site electrostatic potentialsand hydrophobicity for the substrate binding cleft of MAPKAP kinase-2,Akt and Chk1. FIG. 6A is a table showing the optimal substratephosphorylation motifs for Akt/PKB, Chk1, Chk2 and MAPKAP kinase-2. FIG.6B is a ribbons representation of the MAPKAP kinase-2 kinase domain in asimilar orientation as that shown in FIGS. 6C-6E (upper), and in anorthogonal orientation (lower) with stick representations of thesubstrate peptide in the active site. The figure was created usingMolscript and Raster3D. FIG. 6C shows molecular surface representationsof the Akt/PKB active site (PDB code 106K) using GRASP. Electrostaticpotentials (left) and hydrophobicity (right) are indicated by shading.The GSK3 substrate peptide GRPRTTSFAE (SEQ ID NO: 5), with thephospho-acceptor indicated in bold, is shown in stick representation.FIG. 6D shows molecular surface representations of the MAPKAP kinase-2active site (PDB code 1NY3). Electrostatic and hydrophobic potentialsare shaded as in FIG. 6C. The optimal substrate peptide LQRQLSIA (SEQ IDNO: 6) is shown in stick representation. FIG. 6E shows molecular surfacerepresentations of the Chk1 active site (PDB code 1IA8). Electrostaticand hydrophobic potentials are shaded as in FIG. 6C. Stickrepresentation of the modeled Cdc25C substrate peptide (LYRSPSMPL) (SEQID NO: 7) is shown. The region corresponding to the Ser−5, Ser−3 andSer+1 positions of the substrate peptides in FIGS. 6C, 6D, and 6E isindicated by dashed circles.

FIG. 7 is a representation of a unified model of the kinase-dependentDNA damage checkpoint. In this model, parallel pathways in the DNAdamage checkpoint signal transduction network converge on commonsubstrates by signaling to downstream kinases with similarphosphorylation motif specificities. 4 indicates hydrophobic residues.The dashed line from Chk1 to Cdc25B/C indicates that thisphosphorylation event remains controversial in response to ionizingradiation.

In FIG. 8A, HeLa cells were incubated with different chemotherapeuticagents for various times as indicated. Lysates were probed for MAPKAPkinase-2, phospho T334 MAPKAP kinase-2 and β-actin as indicated. In FIG.8B, U2OS cells were incubated with different chemotherapeutic agents forvarious times as indicated. Lysates were probed for MAPKAP kinase-2,phospho T334 MAPKAP kinase-2 and β-actin as indicated. In FIG. 8C, U87MGcells were incubated with different chemotherapeutic agents for varioustimes as indicated. Lysates were probed for MAPKAP kinase-2, phosphoT334 MAPKAP kinase-2 and b-actin as indicated.

In FIG. 9A, U2OS cells were stably transfected with a lentiviraltransfection system. Cells were then treated with cisplatin andnocodazole as indicated. After the incubation, cells were harvested andlabeled with propidium iodide and phospho histone H3 antibodies forsubsequent FACS analysis. In FIG. 9B, U2OS cells were stably transfectedwith a lentiviral transfection system. Cells were then treated withdoxorubicin and nocodazole as indicated. After the incubation, cellswere harvested and labeled with propidium iodide and phospho histone H3antibodies for subsequent FACS analysis.

In FIG. 10, U2OS cells were transiently transfected with siRNA targetingGFP or MAPKAP kinase-2 as indicated. After incubation with cisplatin forthe indicated times, cells were harvested and lysates were blotted forCDC25A, MAPKAP kinase-2 and β-actin as indicated.

FIG. 11A is a graph summarizing the results of two independent platingassays. Transiently transfected cells were treated with cisplatin for 8hours. After the incubation, cells were split 1:20 and re-seeded intonew dishes. After a week, cells were methanol-fixed and stained withmodified Giemsa™ stain for 2 min. Colonies were then counted. FIG. 11Bis a graph summarizing the results of two independent plating assays.Transiently transfected cells were treated with doxorubicin for 8 hours.After the incubation, cells were split 1:20 and re-seeded into newdishes. After a week, cells were methanol-fixed and stained withmodified Giemsa™ stain for 2 min. Colonies were then counted. FIG. 11Cshows photographs of the original plates for FIG. 11A. Insets showhigher magnifications of single colonies. FIG. 11D shows photographs ofthe original plates for FIG. 11B. Insets show higher magnifications ofsingle colonies.

FIGS. 12A-12F show that the MAPKAP kinase-2 pathway is activated afterDNA-damaging chemotherapy. In FIGS. 12A-12D, the kinetics of MAPKAPkinase-2 and p38 MAPK activation are shown. U2OS cells were treated with10 μM cisplatin (FIG. 12A), 10 μM camptothecin (FIG. 12B), 10 μMdoxorubicin (FIG. 12C), or DMSO control (FIG. 12D) for the indicatedtimes. Cell lysates were probed for total and phosphorylated/activatedforms of MAPKAP kinase-2 (MK-2) and p38 MAPK by western blotting.β-actin staining served as a loading control. FIG. 12E shows that MAPKAPkinase-2 activation is p38 MAPK-dependent. U2OS cells were treated withthe p38 MAPK specific inhibitor SB203580 (10 μM) or DMSO vehicle for 30min prior to exposure to chemotherapeutic drugs as in FIGS. 12A-12D.Total and phosphorylated/activated p38 was determined by immunoblottingas above. FIG. 12F shows that activation of MAPKAP kinase-2 parallelsthe formation of γH2AX nuclear foci. U2OS cells were either mock treatedor incubated with cisplatin (10 μM), camptothecin (10 μM) or doxorubicin(10 μM). Cells were immunostained one and four hours later using anantibody against γ-H2AX, and counterstained with DAPI (FIG. 12 F). FIG.12 G is a summary of the requirement for ATM and/or ATR for theactivation of MK2.

FIGS. 13A-13C show that ATM/ATR are required for activation of MAPKAPkinase-2 by DNA damaging drugs. FIG. 13A shows that MAPKAP kinase-2activation by doxorubicin, but not by cisplatin or UV, involves ATM.GM05849 A-T fibroblasts and corresponding control GM00637 fibroblastswere treated with cisplatin (10 μM), doxorubicin (10 μM), UV irradiation(20J/m²) or DMSO (control) for two or eight hours. Cell lysates wereprobed for total and activated MAPKAP kinase-2 by immunoblotting as inFIG. 12, with β-actin as a loading control. FIG. 13B shows that MAPKAPkinase-2 activation by both cisplatin and doxorubicin is ATR-dependent.GM18366 ATR-defective cells from a patient with Seckel syndrome or thecorresponding control GM00023 fibroblasts were treated and analyzed asin FIG. 13A. In contrast to cisplatin and doxorubicin, MAPKAP kinase-2activation after UV did not require ATR. FIG. 13C shows that caffeineinhibits MAPKAP kinase-2 activation by cisplatin and doxorubicin but notUV. U2OS cells were treated with 20 mM caffeine or vehicle alone for 30min prior to exposure to the DNA damaging agents. in FIG. 13A. Celllysates were analyzed for MAPKAP kinase-2 activation as above.

FIGS. 14A-14C show that MAPKAP Kinase 2 mediates a G₂/M arrest followingdoxorubicin treatment. FIG. 14A shows that RNAi down-regulation ofMAPKAP kinase-2 ablates the doxorubicin-induced G₂/M checkpoint. U2OScells stably expressing control luciferase shRNA or MAPKAP kinase-2shRNA were cultured in the absence or presence of 10 μM doxorubicin andcell cycle profiles analyzed thirty hours later by FACS using PI for DNAcontent and phosphohistone H3 staining as an indicator of mitosis. Inthe lower set of panels, nocodazole (100 nM) was added three hoursfollowing doxorubicin addition. Note that in addition to loss of theprominent G₂/M checkpoint, the G₁ and S phase components are alsoeliminated in MK2-depleted cells following doxorubicin+nocodazoletreatment. The efficiency of MK2 depletion was analyzed byimmunoblotting total cell lysates (lower left). FIG. 14B shows thatdown-regulation of MAPKAP kinase-2 does not impair Chk1 activation.Luciferase shRNA- or MAPKAP kinase-2 shRNA expressing U2OS cells weremock treated or exposed to 10 μM doxorubicin for thirty hours. Totalcell lysates were immunoblotted for the presence of MAPKAP kinase-2 andtotal and activated forms of Chk1. FIG. 14C shows that doxorubicin andcamptothecin-induced binding of Cdc25B to 14-3-3 is lost in MAPKAPkinase-2 depleted cells. U2OS cells were mock treated or treated with 10M cisplatin, 10 μM camptothecin, or 10 μM doxorubicin for eight hours.The presence of 14-3-3 binding sites on Cdc25B was monitored byincubating the lysates with bead-bound GST-14-3-3 followed byimmunoblotting of the pulled-down material.

FIGS. 15A-15C show that MAPKAP kinase-2 controls the G₁/S checkpoint inresponse to cisplatin treatment. FIG. 15A shows that RNAidown-regulation of MAPKAP kinase-2 ablates the cisplatin-induced G₁/Scheckpoint. U2OS cells stably expressing control luciferase shRNA orMAPKAP kinase-2 shRNA were cultured in the absence or presence of 10 μMcisplatin and cell cycle profiles analyzed thirty hours later by FACSusing PI for DNA content and phosphohistone H3 staining as an indicatorof mitosis. In the lower set of panels, nocodazole (100 nM) was addedthree hours following cisplatin addition. FIG. 15B shows thatcisplatin-induced reduction of Cdc25A levels is impaired in MAPKAPkinase-2 depleted cells despite activation of Chk1. Luciferase shRNA- orMAPKAP kinase-2 shRNA expressing U2OS cells were mock treated or exposedto 10 μM cisplatin for eight and twelve hours. Total cell lysates wereimmunoblotted for Cdc25A, total MAPKAP kinase-2 and activated Chk1.α-actin was used as a loading control. FIG. 15C shows that MAPKAPkinase-2 phosphorylates Cdc25A in vitro. GST-tagged Cdc25A wasphosphorylated in 30 μl kinase reactions containing either 0.3 μM Chk1or 0.1 μM MAPKAP kinase-2 for 20 min at 30° C. Samples were analyzed bySDS-PAGE autoradiography. Equal substrate loading was assessed byimmunoblotting for GST.

FIGS. 16A-16D show that MAPKAP kinase-2 depletion sensitizes U2OS cellsto the antiproliferative effects of cisplatin and doxorubicin.Luciferase shRNA- or MAPKAP Kinase 2 shRNA-expressing U2OS cells weremock treated or treated for eight hours with increasing doses ofcisplatin (FIG. 16A) or doxorubicin (FIG. 16B) in a clonogenic survivalassay. Cells were washed, trypsinized, and replated at a density of 5000cells/10 cm² dish. Eight days later, colonies were visualized usingCrystal Violet staining and counted. Insets show magnified views. FIGS.16C and 16D are graphs showing the quantitation of the results shown inFIGS. 16A and 16B. Assays were performed in triplicate for eachcondition and normalized to mock treated cells.

FIGS. 17A-17D show that MAPKAP kinase-2 depletion suppresses tumorformation in vivo after implantation of chemotherapy-treated cancercells. H-Ras-V12 transformed p53^(−/−) MEFs were transfected with siRNAoligonucleotides against GFP or MAPKAP kinase-2. Forty-eight hoursfollowing transfection, cells were treated for eight hours with eithercisplatin (1 μm), doxorubicin (0.1 μm), or vehicle alone. Two individualinjections of 10⁶ cells each were performed into the subcutaneoustissues of each flank of NCR nude outbred mice. FIG. 17A is a dorsalview of the resulting tumors fifteen days after each of the indicatedtreatments. Control siRNA-transfected cells are in the right flank andMAPKAP kinase-2 siRNA-treated cells are in the left flank. FIG. 17B is aclose-up view of the resulting tumors that formed in the absence of DNAdamaging chemotherapy pre-treatment. In FIG. 17C, the efficiency ofsiRNA-mediated knockdown on murine MAPKAP kinase-2 was assessed byimmunoblotting lysates from the MEFs prior to tumor implantation. FIG.17D is a graph showing an analysis of tumor weight at the fifteen-dayendpoint.

FIGS. 18A-18D show that MAPKAP kinase-2 depletion enhances regression ofestablished tumors after DNA damaging chemotherapy in a murine model. InFIG. 18A, H-Ras-V12 transformed p53^(−/−) MEFs were infected withlentiviruses encoding U6 promoter-driven luciferase shRNA or MAPKAPkinase-2 shRNA, and CMV promoter-driven GFP. Three days post-infection,GFP expressing cells were selected by FACS and cultured for anadditional 7 days. Efficiency of MAPKAP kinase-2 knockdown in the entireGFP-positive population was then assessed by immunoblotting of totalcell lysates. In FIG. 18B, following subcutaneous injection of 10⁶ cellsinto the flanks of NCR nude outbred mice as in FIG. 17, tumor growth wasmeasured every two days. The arrow indicates the start ofintraperitoneal administration of DMSO, cisplatin, or doxorubicin on daytwelve. In the absence of DNA damaging chemotherapy, the MAPKAP kinase-2depleted tumors were statistically significantly larger than the controltumors at each time point beginning on day thirteen (Student's t-test,2-tailed, p<0.02). In contrast, after cisplatin or doxorubicin treatmentthe MAPKAP kinase-2 depleted tumors were statistically smaller than thecontrol tumors beginning on days twenty-one and twenty-three,respectively (p<0.02). In FIG. 18C, the upper panels are dorsal views ofthe tumors in situ fourteen days after initiation of the indicatedtreatments, corresponding to twenty-six days after tumor cellimplantation. Middle panels are corresponding fluorescence images. Lowerpanels are close-up views of the excised tumors. FIG. 18D is a graphshowing an analysis of tumor weight at the twenty-six-day endpoint.

FIG. 19 shows that cisplatin and doxorubicin activate MAPKAP kinase-2independently of Chk1. U2OS cells were transfected with siRNAoligonucleotides targeting GFP or Chk1. Forty eight hours followingtransfection, cells were treated with 10 μM cisplatin or 10 μMdoxorubicin for twelve hours, lysed, and probed for levels of total andphosphorylated/activated MAPKAP kinase-2. The efficiency of theknockdown was assessed by immunoblotting for Chk1.

FIGS. 20A-20E show that overexpression of Chk1 rescues the loss of G₁/Sand G₂/M checkpoints and enhances resistance to genotoxic stress inMAPKAP kinase-2-depleted cells. In FIGS. 20A-20B, luciferase and MAPKAPkinase-2 shRNA expressing U2OS cells were transiently transfected withhuman Chk1 or empty vector alone. Cells were then exposed to 10 μMcisplatin (FIG. 20A) or 10 μM doxorubicin (FIG. 20B). To arrest cyclingcells in mitosis, 100 nM nocodazole was added to the media after threehours of treatment where indicated. The cell cycle profile was analyzedafter thirty hours of treatment by FACS using PI for DNA content andphospho-histone H3 staining. In FIGS. 20C-20E, the cell types in FIGS.20A and 20B above were used in clonogenic survival assays as in FIG. 17.Cells were treated with increasing doses of cisplatin (FIG. 20C) ordoxorubicin (FIG. 20D) for eight hours, or mock irradiated or UVirradiated (20J/m²) (FIG. 20E), washed, trypsinized and seeded at adensity of 5000 cells/10 cm² dish. The number of surviving colonies wasquantitated eight days later. Assays were performed in triplicate foreach condition and normalized to mock treated cells.

FIGS. 21A-21D show that UCN-01 potently inhibits MAPKAP kinase-2. InFIG. 21A, in vitro kinase assays in the presence of increasing doses ofUCN-01 were performed with Chk1 and MAPKAP kinase-2 using the MK-2tideas a substrate. FIG. 21B shows the structural basis for UCN-01inhibition of MAPKAP kinase-2. Ribbon diagrams and molecular surfaces ofthe Chk1:UCN-01 complex (panels 1, 4) and the MAPKAPkinase-2:staurosporine complex with UCN-01 are modeled ontostaurosporine (panels 2, 3, 5). Panel 3 is rotated 90° from the view inpanel 2. The arrow points to the unique 7-hydroxy group of UCN-01 withVan der Waals radii indicated by dots. FIG. 21C shows that UCN-01inhibits MAPKAP kinase-2 in U2OS cells. Luciferase shRNA- or MAPKAPKinase 2 shRNA-expressing cells were incubated at 37° C. or 42° C. fortwo hours in the absence or presence of 200 nM UCN-01. Cells were lysedand probed for total hsp-27, hsp-27 pS82, and MAPKAP kinase-2 byimmunoblotting. FIG. 21D shows that UCN-01 inhibition of hsp-27 isindependent of Chk1. GFP or Chk1 siRNA-transfected cells were incubatedat 42° C. or 37° C. for two hours in the absence or presence of 200 nMUCN-01. Cells were then lysed and probed for total hsp-27, hsp-27 pS82,and Chk1 by immunoblotting.

FIG. 22 is a representation of a model for MAPKAP kinase-2 checkpointsignaling in response to DNA damaging chemotherapy. Checkpoint functionin response to DNA damaging agents normally requires the combined actionof both the Chk1 and MAPKAP kinase-2 pathways, and both pathways aresimultaneously inhibited by the indolocarbazole drug UCN-01.

FIGS. 23A-23B show that camptothecin activates MAPKAP kinase-2 in anATR-dependent and ATM-independent manner. In FIG. 23A, GM18366ATR-defective cells from a patient with Seckel syndrome or thecorresponding control GM00023 fibroblasts were treated with camptothecin(10 μM) for two or eight hours. Cell lysates were probed for total andactivated MAPKAP kinase-2 by immunoblotting, and with anti-α-actin as aloading control. In FIG. 23B, GM05849 A-T fibroblasts and correspondingcontrol GM00637 fibroblasts were treated and analyzed as in FIG. 23A.

FIGS. 24A-24D show that treatment with cisplatin and doxorubicinpreferentially induce different cell cycle checkpoints. In FIG. 24A, thecell cycle profile of asynchronous control U2OS cells was analyzed byFACS using PI for DNA content and phospho-histone H3 staining. FIG. 24Bshows that cells treated with doxorubicin (10 μM) for eighteen hourspreferentially accumulate at the G₂/M boundary, with a smaller componentin G₁ and S. FIG. 24C shows that cells treated with cisplatin (10 μM)for eighteen hours preferentially accumulate in G₁/S with a smallcomponent at G₂/M. FIG. 24D shows that cells treated with nocodazole(100 nM) for eighteen hours accumulate in M with ˜42% staining stronglyfor phospho-histone H3.

FIG. 25 shows the effect of overexpression of Chk1 in control and MAPKAPkinase-2 knockdown U2OS cells. U2OS cells stably expressing luciferaseor MAPKAP kinase-2 shRNA were transiently transfected with FLAG-taggedChk1 in the expression vector pHURRA, or with vector alone. Cell lysateswere probed for total MAPKAP kinase-2 and Chk1 levels, with β-actin as aloading control.

FIG. 26 shows that overexpression of Chk1 rescues the loss of theUV-induced G₁/S checkpoint. Luciferase and MAPKAP kinase-2 shRNAexpressing U2OS cells were transiently transfected with human Chk1 orempty vector alone. Cells were then irradiated with 20 J/m² of UV light.To arrest cycling cells in mitosis, 100 nM nocodazole was added to themedia after three hours of treatment where indicated. The cell cycleprofile was analyzed after thirty hours of treatment by FACS using PIfor DNA content and phospho-histone H3 staining. In addition to theprominent G₁/S checkpoint, a minor G₂/M checkpoint was also observed inthe control cells, but not the MAPKAP kinase-2 depleted cells, afterirradiation, and this checkpoint was also restored upon overexpressionof Chk1.

FIG. 27 shows that Chk1 overexpression rescues clonogenic survival inMAPKAP kinase-2 depleted cells treated with cisplatin. U2OS cells stablyexpressing luciferase or MAPKAP kinase-2 shRNA were transientlytransfected with FLAG-tagged Chk1 in the expression vector pHURRA, orwith vector alone. Cells were treated with increasing doses of cisplatinfor eight hours, washed, trypsinized and seeded at a density of 5000cells/10 cm² dish. Surviving colonies were stained with crystal violetand counted eight days later.

FIG. 28 shows that Chk1 overexpression rescues clonogenic survival inMAPKAP kinase-2 depleted cells treated with doxorubicin. U2OS cells asin FIG. 27 were treated with increasing doses of doxorubicin andanalyzed as described above.

FIGS. 29A-29B are graphs showing the results of a colony survival assayin which mouse embryonic fibroblasts (MEFs) were treated with increasingdoses of doxorubicin or cisplatin for eight hours, rinsed twice with PBSand once with media, and re-plated at an initial density of 5,000cells/10 cm² dish. After eleven days, the number of colonies on theplate was counted and normalized to the number of colonies formed by thesame cell type in the absence of treatment with chemotherapeutic drugs.RNA interference using short hairpin RNAs was used with both the p53wild-type MEFs and the p53^(−/−) MEFs to knock down the levels of MAPKAPkinase-2 (MK2 shRNA). Short hairpin RNAs against luciferase (luciferaseshRNA) were used as a control. Loss of MAPKAP kinase-2 activity resultedin increased sensitivity to both doxorubicin and cisplatin (i.e.decreased survival after treatment) only in the MEFs that lacked p53.FIG. 29C is a series of Western blots using the indicated antibodies.These blots verify that p53 polypeptide, and the known p53-responsivegene product p21, is induced by DNA damage only in the p53 wild-typecells (lanes 2, 3, 5, and 6), but not in the p53^(−/−) cells (lanes7-12). The blot also verifies that the short hairpin RNA against MAPKAPkinase-2 knocked down the levels of MAPKAP kinase-2 in the MK-2 shRNAtreated cells (lanes 4-6 and 10-12). This also prevented MAPKAP kinase-2activation in the MAPKAP kinase-2 shRNA-treated cells after DNA-damagingchemotherapy, as shown by the lack of phospho-MAPKAP kinase-2 in lanes5-6 and 11-12, compared with the presence of phospho-MAPKAP kinase-2 inthe luciferase shRNA treated cells after DNA-damaging chemotherapy(lanes 2-3 and 8-9).

FIGS. 30A-30D represent pS3WT/WT and p53−/− MEFs stably expressing RNAihairpins against luciferase (FIGS. 30A and 30C) or MK2 (FIG. 30B, and30D). Cells were then treated with low dose cisplatin (1.01M) ordoxorubicin (0.1 μM) for thirty hours, fixed and stained with antibodiesagainst phosphohistone H3, γ-H2AX and cleaved caspase-3. Positivestaining for γ-H2AX in combination with phospho-histone H3 and cleavedcaspase-3 labeling is indicative of mitotic catastrophe, was onlyobserved in MK2 depleted p53−/− cells (FIG. 30D, arrows). Arrowhead inthat panel shows a γ-H2AX-positive cell that does not stain for eitherphospho-histone H3 or cleaved caspase-3. Scale bar, 5 μm.

FIGS. 31A-31C are histograms and western blot images demonstratingMAPKAP Kinase 2 mediates a G₂/M arrest following doxorubicin treatment.RNAi down-regulation of MK2 ablates the doxorubicin-induced G₂/Mcheckpoint (FIG. 31A). p53−/− MEFs stably expressing control luciferaseshRNA (FIG. 31A left panels) or MK2 shRNA (FIG. 31A right panels) werecultured in the absence or presence of 10 μM doxorubicin and cell cycleprofiles analyzed thirty hours later by FACS using PI for DNA content(blue) and phospho-histone H3 staining as an indicator of mitosis (red).In the lower set of panels, nocodazole (100 ng/ml) was added three hoursfollowing doxorubicin addition. Note that in addition to loss of theprominent G₂/M checkpoint, the G₁ and S phase components are alsoeliminated in MK2-depleted cells following doxorubicin+nocodazoletreatment. Down-regulation of MK2 does not impair Chk1 activation (FIG.31B). Luciferase shRNA or MK2 shRNA expressing p53−/− MEFs were mocktreated or exposed to 10 μM doxorubicin or 10 μM camptothecin for thirtyhours. Total cell lysates were immunoblotted for the presence of MK2 andtotal and activated forms of Chk1. Doxorubicin and camptothecin-inducedbinding of Cdc25B to 14-3-3 is lost in MK2 depleted cells (FIG. 31C).p53−/− MEFs cells either expressing a luciferase hairpin (upper panel)or a MK2 specific hairpin (lower panel) were mock treated or treatedwith 10M camptothecin (cam) or 10 μM doxorubicin (dox) for eight hour.The presence of 14-3-3-binding sites on Cdc25B was monitored byincubating the lysates with bead-bound GST-14-3-3 β/ζ followed byimmunoblotting of the pulled-down material.

FIGS. 32A-32B are histograms and western blot images demonstrating MK2controls the S-phase checkpoint in response to cisplatin treatment. RNAidown-regulation of MK2 ablates the cisplatin-induced S-phase checkpoint(FIG. 32A). p53−/− MEFs stably expressing control luciferase shRNA (FIG.32A, left panels) or MK2shRNA (FIG. 32A, right panels) were cultured inthe absence or presence of 10 μM cisplatin and cell cycle profilesanalyzed thirty hours later by FACS using PI for DNA content (blue) andphospho-histone H3 staining as an indicator of mitosis (red). In thelower set of panels, nocodazole (100 ng/ml) was added three hoursfollowing cisplatin addition. Cisplatin-induced degradation of Cdc25A isimpaired in MK2 depleted cells despite activation of Chk1 (FIG. 32B).Luciferase shRNA- or MK2 shRNA expressing p53−/− MEFs were mock treatedor exposed to 10M cisplatin for eight and twelve hours. Total celllysates were immunoblotted for Cdc25A, total MK2 and activated Chk1.β-actin was used as a loading control.

FIGS. 33A-33B are models for re-wiring of cell cycle checkpoint pathwaysin p53-proficient and deficient cells. Checkpoint function inp53-proficient cells is mediated primarily through a robust, sustainedp53 response downstream of ATM, together with Chk1 (FIG. 33A). Althoughnot shown explicitly in the diagram, Chk1 also directly phosphorylatesp53 (Shieh et al., 2000). Under these conditions the presence of MK2 isnot required for cell survival after exposure to DNA damaging agents. Inp53-deficient cancer cells (FIG. 33B), checkpoint signaling followingexposure to DNA damaging agents is mediated through the combined actionof both the Chk1 and the p38 MAPK/MK2pathways. In this situation thep38MAPK/MK2 branch of checkpoint signaling becomes essential for cellsurvival after DNA damage. Both pathways are simultaneously inhibited bythe indolocarbazole drug UCN-01.

DETAILED DESCRIPTION OF THE INVENTION

The invention features methods and compounds that are useful in treatingcellular proliferative disorders. The methods of treatment featureadministration of a compound that is capable of inhibiting an activityof a MAPKAP kinase-2 polypeptide, or a substrate-binding fragmentthereof. Such compounds include, without limitation, compounds thatcontain peptides, peptidomimetics, or nucleic acid molecules. Theinvention further features screening assays for identifying MAPKAPkinase-2 inhibitors. In addition, the invention includes pharmaceuticalcompositions and compounds, e.g., peptides and peptidomimetics, thattarget the substrate-binding site of MAPKAP kinase-2, thereby inhibitingit.

It was recently shown that, in addition to the ATR-Chk1 pathway, the p38SAPK pathway is also required for full activation of the DNA damageresponse following UV irradiation. We now demonstrate that MAPKAPkinase-2, a direct downstream target of p38 SAPK, is directlyresponsible for phosphorylating Cdc25B and C and maintaining the G₁, S,and G₂/M checkpoints in response to UV-induced DNA damage. Thus, MAPKAPkinase-2 constitutes a third checkpoint kinase, in addition to Chk1 andChk2, involved in coordinating the DNA damage response of highereukaryotic cells.

A number of important questions regarding this third DNA damage responsepathway have not been previously answered. Is p38 MAPK/MAPKAP kinase-2activation after DNA damage dependent on ATR or ATM? Is p38 MAPK/MAPKAPkinase-2 cascade important for DNA damage checkpoints in response toother types of genotoxic stress besides UV? How are signals from theChk1 pathway and the MAPKAP kinase-2 pathway integrated together at thesystems level? We were particularly interested in investigating whetherMAPKAP kinase-2/Chk3 participates in the genotoxic stress response ofcells exposed to conventional anti-cancer chemotherapeutic agents. Ademonstration that MAPKAP kinase-2 has an important role in preventingcells with chemotherapy-induced DNA damage from progressing through thecell cycle would implicate MAPKAP kinase-2 as a clinically importanttarget for anti-cancer drug design.

Defining the Optimal Phosphorylation Motif for p38 SAPK

To identify substrates and targets of the p38 SAPK signaling pathwayinvolved in DNA damage responses, we determined the optimal substratephosphorylation motif for p38ax SAPK using oriented peptide libraryscreening. Efficient peptide phosphorylation by p38 SAPK required afixed Pro residue in the Ser+1 position, consistent with the knownidentification of p38 SAPK as a Pro-directed MAP kinase. Screeningperformed with a library containing the degenerate sequence XXXXSPXXXX(SEQ ID NO: 8) (X denotes all amino acids except Cys, Ser, Thr, and Tyr)displayed strongest selection for Pro in the Ser−2 position with weakerselection for other aliphatic residues (FIG. 1A). Additional selectionwas also observed at the Ser−3, Ser−1, and Ser+2 positions.

To further refine the optimal phosphorylation motif, a secondary screenwas performed based on results from the initial screen by using alibrary with Pro fixed in both the Ser−2 and Ser+1 positions, and Ser,Thr, and Tyr included in the X positions. This revealed selection forGln, Met, and Gly in the Ser−1 position, along with slightly weakerselection for Pro, Ser and Thr (FIG. 1A). Gly was the preferred residuein the Ser−3 position, along with Ile, Val, and Tyr. Hydrophobicresidues, particularly aromatic and β-branched amino acids, wereselected at the Ser+2 position. The resulting optimal motif for p38αSAPK determined by oriented peptide library screening closely matchesthe sequence of mapped p38 MAPK phosphorylation sites on most, thoughnot all, known substrates (FIG. 1A).

A peptide containing the optimal p38 SAPK consensus phosphorylationmotif GPQSPI (SEQ ID NO: 9), “p38tide,” was synthesized for kineticanalysis. This peptide was readily phosphorylated by p38 SAPK in vitro;however, it failed to display saturable Michaelis-Menton-type kinetics(FIG. 1B). Instead, the initial velocity increased linearly withincreasing p38tide concentration up to 1400 μM. This finding suggeststhat additional interactions besides an optimal phosphorylation motifare likely to be involved in optimizing p38 SAPK-substrate binding, suchas MAP kinase docking sites.

To search for potential p38 SAPK substrates, particularly those relevantto DNA damage signaling, the Swiss-Prot database was queried with thep38 SAPK consensus phosphorylation motif using Scansite. Other thanGADD153, a known p38 SAPK substrate, we were unable to identify any DNAdamage response proteins in the top 250 hits. Database searching did,however, reveal two tandem near-optimal p38 SAPK phosphorylation sites(Ser345 and Ser348) in p47phox, a cytosolic component of the NADPHoxidase enzyme. A peptide containing this sequence, PGPQSPGSPL (SEQ IDNO: 10), “p47tide,” was strongly phosphorylated by p38 SAPK, but likep38tide, the isolated peptide displayed linear non-saturable kinetics(FIG. 1B).

Wild-type and mutant versions of GST-tagged full-length p47phox protein,rather than isolated peptides, were then used as substrates for in vitrophosphorylation reactions. The wild-type full-length p47phox protein wasrapidly phosphorylated by p38a SAPK (FIGS. 1C and 1D). Mutation ofSer345-Ala had a more pronounced effect on p47phox phosphorylation thanmutation of Ser348-Ala, in excellent agreement with the observation thatthe Ser345 site is a better match for the optimal p38 SAPK consensusmotif than the Ser348 site. Simultaneous mutation of both Ser345 andSer348 to Ala eliminated phosphorylation of p47phox by p38 SAPKaltogether. Kinetic analysis revealed classical Michaelis-Mentonbehavior for p38 SAPK phosphorylation of the wild-type p47phox with aK_(m) of 6.0 μM and a V_(max) of 36.6 nmol/min/μg. Mutation of Ser345 toAla both increased the K_(m) and reduced the V_(max), while mutation ofSer348 to Ala primarily increased the K_(m) (FIG. 1E).

These data from isolated peptides and intact proteins argue thatefficient substrate phosphorylation by p38 SAPK requires sequences withreasonable matches to the optimal substrate motif determined by orientedpeptide library screening, and that additional interactions involvingMAPK docking sites are likely to be critical for strong kinase-substrateinteractions. Several docking motifs have been identified for p38 SAPK,particularly a short cluster of positively charged amino acid residuesoften flanked by hydrophobic amino acids. Two sequences corresponding tothis type of docking motif are present near the p38SAPK phosphorylationsites in p47 phox, 1HQRSRKRLSQ (SEQ ID NO: 11) and VRFLQQRRRQA (SEQ IDNO: 12). Mutation of RRR to LLL in the latter motif decreased the rateof p38α SAPK phosphorylation of p47phox by over 70%.

Bulavin et al. (Nature, 411:102-107, 2001) implicated p38 SAPK in theDNA damage response pathway and reported that p38 SAPK was directlyresponsible for generating a 14-3-3-binding site on Cdc25B (Ser323 inCdc25B2; Ser309 in Cdc25B1) in response to UV-C-induced DNA damage. Likep47phox, Cdc25B contains a potential p38 SAPK docking motif, PVQNKRRRSV(SEQ ID NO: 13); however, the sequence flanking Ser323, LXRSPSMP (SEQ IDNO: 14), lacks a Pro in the Ser+1 position and does not resemble theoptimal p38 SAPK motif shown in FIG. 1A. As shown in FIG. 2A,recombinant p38 SAPK readily phosphorylated bacterially produced Cdc25Bin vitro. However, this phosphorylation did not induce 14-3-3-binding,and a Ser323→Ala mutant form of Cdc25B was phosphorylated by p38 SAPKequivalently to the wild-type Cdc25B protein. These data argue that,while Cdc25B may be a p38 SAPK substrate, this phosphorylation event isnot responsible for the 14-3-3-binding event that results in a G₂/Mcheckpoint.

Defining the Optimal Phosphorylation Motif for MAPKAP Kinase-2

A number of Ser/Thr kinases are activated downstream of p38 SAPK,including MAPKAP Kinases-2 and -3, MNK1 and MNK2, MSK1 and MSK2, andPRAK. In response to UV-B-induced DNA damage, She et al. (Oncogene,21:1580-1589, 2002) reported that MAPKAP kinase-2 could phosphorylatep53 on Ser20, the same site that is phosphorylated by twowell-established checkpoint kinases, Chk1 and Chk2. Both Chk1 and Chk2can also phosphorylate Cdc25 family members to create 14-3-3 bindingsites, suggesting that MAPKAP kinase-2 might share a similar motif. Theoptimal substrate phosphorylation motif for MAPKAP kinase-2 wastherefore investigated using oriented peptide library screening.

Efficient peptide phosphorylation by MAPKAP kinase-2 was only observedwith a library containing a fixed Arg in the Ser−3 position(XXXXRXXSXXXX (SEQ ID NO: 15), where X denotes all amino acids exceptCys, Ser, Thr, or Tyr). A critical step in determining protein kinasephosphorylation motifs by peptide library screening involvespurification of the phosphorylated peptides from the non-phosphorylatedpeptide background. In the case of MAPKAP kinase-2, this wasdramatically improved by conversion of all Glu and Asp residues to theirmethyl esters prior to metal-affinity chromatography and sequencing.This resulting motif revealed clear amino acid selection at almost alldegenerate positions (FIG. 3A). MAPKAP kinase-2 displayed strongselection for the hydrophobic residues Leu, Phe, Ile, and Val in theSer−5 position and the Ser+1 position. Strong selection was alsoobserved for Gln in the Ser−2 position, and modest selection for Leu inthe Ser−1 position. The motif determined for MAPKAP kinase-2 usingoriented peptide library screening is in remarkably good agreement withthe sequence of mapped MAPKAP kinase-2 phosphorylation sites on knownsubstrates (FIG. 3A, bottom), which primarily contain Leu, Ile or Phe inthe Ser−5 position; Arg in the Ser−3 position; Gln, Ser, or Thr in theSer−2 position; Leu, Val or Pro in the Ser−1 position; and hydrophobicresidues along with Glu in the Ser+1 position. The preference for polarresidues Ser and Thr in addition to Gln in the Ser−2 position in knownMAPKAP kinase-2 substrates would not have been detected by orientedpeptide library screening, since Ser and Thr were not present in thelibrary.

To verify the peptide library screening results, individual peptides(MK2tides) containing the optimal MAPKAP kinase-2 consensus motifLQRQLSI (SEQ ID NO: 16), or mutant versions with Ala substituted at eachposition in the motif, were synthesized and used for kinetic analysis(FIGS. 3B and 3C). The optimal MK2tide displayed a K_(m) value two-foldlower than the best MAPKAP kinase-2 peptide substrate known to date, asequence derived from HSP27. Substitution of Ala at each position in themotif affected K_(m) and V_(max) differently, with some positionsshowing primarily a K_(m) effect (i.e., Arg in the Ser−3 position),while others revealed a primary effect on V_(max) (i.e., Leu in theSer−5 position) (FIG. 3C). The rank order of importance of key residuesis Arg-3>Leu-5≈Ile+1>Gln-3. The optimal MK2tide had neither the lowestK_(m) nor the highest V_(max), but rather had the highest V_(max)/K_(m)ratio, consistent with the fact that the peptide library screeningapproach selects substrates on the basis of optimal V_(max)/K_(m),rather than low K_(m) or high V_(max) alone. Combining the data fromoriented peptide library screening, known substrate sequences, and ourkinetic studies, the optimal MAPKAP kinase-2 phosphorylation motif is[L/F/I]XR[Q/S/T]L[S/T][Hydrophobic] (SEQ ID NO: 17).

The optimal MAPKAP kinase-2 substrate motif is an excellent match forthe known Ser323 phosphorylation/14-3-3 binding motif in Cdc25B, as wellas the Ser216 phosphorylation/14-3-3-binding site in Cdc25C (FIG. 3A).Initial experiments focused on Cdc25B, since, unlike Cdc25C, Cdc25B canbe produced in modest quantities in bacteria, and the Ser323 site inCdc25B had been previously reported to be a direct p38 SAPK site.Incubation of recombinant Cdc25B with purified MAPKAP kinase-2 resultedin significant Cdc25B phosphorylation and strong binding of thephosphorylated protein to 14-3-3 (FIG. 2A). Mutation of Ser323→Alasubstantially reduced the ability of MAPKAP kinase-2 to phosphorylateCdc25B, and completely eliminated the ability of Cdc25B to bind to14-3-3 (FIG. 2A). These in vitro results strongly suggest that MAPKAPkinase-2 is the critical Cdc25/14-3-3 checkpoint kinase downstream ofDNA damage signals relayed by the p38 SAPK pathway.

MAPKAP Kinase-2 is Critical for the G₂/M Checkpoint following UV-InducedDNA Damage

The importance of MAPKAP kinase-2 in DNA damage checkpoint function wasinvestigated in U2OS cells. Activation of MAPKAP kinase-2 in response toUV-C irradiation-induced DNA damage (FIG. 4A) was monitored by itsreduced mobility on SDS-PAGE gels, and by immunoblotting using aphospho-specific antibody against pThr344, a site phosphorylated by p38and required for MAPKAP kinase-2 activation. As shown in FIG. 2B, MAPKAPkinase-2 was activated within one hour of irradiation, and remainedactivated for the eight hour duration of the experiment. The kinetics ofMAPKAP kinase-2 activation paralleled the ability of Cdc25B from thesecells to bind to 14-3-3. Based on these data, a two hour time point waschosen for use in further studies.

RNA interference was used to confirm a direct role for endogenous MAPKAPkinase-2 in the UV-induced DNA damage response. Treatment of U2OS cellswith MAPKAP kinase-2-specific siRNA oligonucleotides, but not withcontrol GFP siRNA oligonucleotides, resulted in a substantial reductionof MAPKAP kinase-2 to nearly undetectable levels by forty-eight hoursafter transfection (FIG. 2C). No reduction in the levels or UV-C-inducedactivation of p38 SAPK, Chk1 or Chk2 was observed in these cells.Despite the presence of these other active kinases, siRNA-mediatedknockdown of MAPKAP kinase-2 caused a loss of both Cdc25B- andCdc25C-binding to 14-3-3 after UV-C exposure (FIG. 2C).

We studied cell cycle progression in the control GFP and MAPKAP kinase-2knockdown cells following UV-C-irradiation using FACS (FIGS. 4A-4K). Inthese experiments, cells were irradiated with 20 J/m² of UV-C radiation,allowed to recover for two hours, then placed in nocodazole-containingmedia for an additional sixteen hours to cause any cells progressingthrough the cell cycle to arrest in mitosis, where they can stained forthe mitotic marker phosho-histone H3. Under these conditions,un-irradiated cultures of asynchronous GFP siRNA-transfected cellsaccumulated in a 4N-DNA-containing peak, with prominent levels ofphospho-histone H3 staining (FIGS. 4B and 4C), consistent with anocodazole-mediated M-phase arrest. In response to UV-irradiation,control cells displayed a prominent G₁, S, and G₂ distribution, withnear-complete loss of phosphohistone H3 staining, indicating intact G₁,S, and G₂ checkpoints (FIGS. 4D and 4E).

The behavior of the MAPKAP kinase-2 siRNA transfected cells wasdramatically different. In the absence of UV irradiation, MAPKAPkinase-2 siRNA transfected cells, like control GFP siRNA-transfectedcells, accumulate in a 4N DNA-containing peak with high levels ofphospho-histone H3 staining (FIGS. 4F and 4G). Following UV-induced DNAdamage, however, the MAPKAP kinase-2 knockdown cells failed to arrestcell cycle progression. Instead, these cells proceeded to enter mitosisto the same extent as unirradiated cells, as shown by a comparable4N-DNA peak and similar levels of phoshohistone H3 staining as thoseobserved in un-irradiated cells (FIGS. 4H and 41). Together with theCdc25B/C: 14-3-3 results in FIG. 2C, these FACS data demonstrate thatMAPKAP kinase-2 is critical for the UV-induced G₂/M checkpoint inresponse to UV-irradiation. In contrast to the UV response, summarizedin FIG. 4J, the G₂/M checkpoint response to ionizing radiation in MAPKAPkinase-2 knockdown cells is intact (FIG. 4K).

MAPKAP Kinase-2 is Critical for the S-Phase Checkpoint and G₁ ArrestFollowing UV-Induced DNA Damage

The MAPKAP kinase-2 knockdown cells in FIGS. 4A-4K also showed a loss ofthe G₁ and S-phase checkpoints following DNA damage, sinceUV-irradiation of asynchronous cultures resulted in accumulation of thecells in a 4N DNA-containing peak when the cells were transferred tonocodazole-containing medium. To investigate the direct role of MAPKAPkinase-2 in S-phase checkpoint function, control or MAPKAP kinase-2knockdown U2OS cells were UV-irradiated, allowed to recover for 30 min,and then labeled with BrdU for various times. In the absence ofirradiation, 42% of the control siRNA-transfected cells showedsubstantial BrdU incorporation after twelve hours, compared with 53% ofthe MAPKAP kinase-2-siRNA transfected cells (FIGS. 5A and 5B). When thecells were irradiated with 20 J/m² of UV light prior to BrdU labeling,only 3.5% of the control siRNA transfected cells showed BrdUincorporation at twelve hours. In marked contrast, 48% of the MAPKAPkinase-2-knockdown cells continued to incorporate substantial amounts ofBrdU. A similar difference in BrdU uptake between control siRNA-treatedcells and MAPKAP kinase-2-knockdown cells was also seen at shorter timesafter irradiation (FIG. 5B).

Examination of the FACS profiles twelve hours following UV-irradiationrevealed a dramatic decrease in the G₁ population in the MAPKAPkinase-2-knockdown cells compared with the control GFP siRNA-transducedcells (FIG. 5A, right-most upper and lower FACS profiles). This loss ofthe G₁ peak, together with the increased percentage of cells showingBrdU incorporation at twelve hours versus two hours of labeling, impliesthat endogenous MAPKAP kinase-2 plays important roles in both theinhibition of DNA synthesis following damage (S-phase checkpointfunction), and in the damage-induced arrest of cells in G₁ prior toS-phase entry (G₁/S checkpoint function). Loss of the G₁/S and S-phasecheckpoints in MAPKAP kinase-2 knockdown cells was associated withhigher levels of Cdc25A, decreased levels of p53, and reducedphosphorylation of p53 on Ser20 following UV-irradiation compared withcontrol siRNA-treated cells (FIG. 2C).

The fate of S-phase control or MAPKAP kinase-2 siRNA-treated cells inresponse to UV-C-induced DNA damage was examined by using FACS. In thisexperiment, asynchronous cells were mock-treated or irradiated with 20J/m² of UV-C radiation and then pulse-labeled with BrdU. The cellsshowing BrdU uptake were subsequently analyzed ten and twenty hourslater (FIG. 5C). In both non-irradiated control and MAPKAP kinase-2knockdown cells, the BrdU pulse-labeled population showed a late S andG₂/M distribution at ten hours, and a re-appearance of a G₁ peak attwenty hours, indicating full transit through the cell cycle. Inresponse to UV-C irradiation, control siRNA-treated cells failed to showsignificant BrdU uptake upon which to gate for FACS analysis (FIG. 5C,lower left panel). In contrast, the large population of MAPKAP kinase-2siRNA treated cells, which had lost the S-phase checkpoint andincorporated BrdU, went on to display a greatly reduced G₁ peak attwenty hours, with many cells showing DNA staining >4N (FIG. 5C, bracketin lower right panel), consistent with mitotic death and exit from thecell cycle.

MAPKAP Kinase-2 Depleted Cells are more Sensitive to DNA Damage-InducedCell Death

The experiments in FIGS. 4 and 5A-5C indicate that MAPKAP kinase-2 isinvolved in each of the cell cycle checkpoints triggered by UV-inducedDNA damage. To determine the effect of MAPKAP kinase-2 depletion on cellsurvival, we transfected cells with control siRNA or MAPKAP kinase-2siRNA for forty-eight hours, trypsinized, replated, and analyzed forcolony formation in response to various doses of UV-C irradiation twelvehours after re-plating. As shown in FIGS. 5D and 5E, MAPKAP kinase-2knockdown cells displayed a significant reduction in colony formationwhen compared to control-treated cells at all doses of UV-C irradiationexamined. This difference in survival after UV-C exposure was mostpronounced at low to moderate UV doses.

A Structural Model for MAPKAP Kinase-2 Substrate Selectivity.

The optimal phosphorylation motif determined for MAPKAP kinase-2 isstrikingly similar to that determined for two other checkpoint kinases,Chk1 and Chk2 (FIG. 6A). All three of these CAMK superfamilymembers—MAPKAP kinase-2, Chk1, and Chk2—strongly select for aliphaticresidues in the Ser−5 position, Arg in the Ser−3 position, andaromatic/aliphatic residues in the Ser+1 position, along with additionalless stringent selection for particular amino acids in other positions(FIG. 6A). In contrast, members of the AGC kinase superfamily, such asAkt/PKB and conventional protein kinase C superfamily members,preferentially phosphorylate sequences containing Arg residues in boththe Ser−5 and Ser−3 positions, and play important roles inanti-apoptotic signaling and other signaling events unique todifferentiated cell function, rather than critical roles in cell cyclecontrol.

To investigate the structural basis for substrate motif selection, weperformed molecular modeling studies of activated MAPKAP kinase-2, usingthe published MAPKAP kinase-2:ADP co-crystal structure (Underwood etal., Structure, 11:627-636, 2003) as a base model. The optimal substratepeptide LQRQLSIA (SEQ ID NO: 6) was modeled into the kinase active sitein an extended conformation (FIG. 6D), and the kinase:substrate complexcompared with the structures of Akt/PKB:AMP-PNP:GSK3-peptide ternarycomplex (Yang et al., Nat. Struct. Biol., 9:940-944, 2002) (FIG. 6C) andthe Chk1 crystal structure containing a modeled Cdc25C peptide (Chen etal., Cell, 100:681-692, 2000) (FIG. 6E). Strong selection for Arg in theSer−3 position for MAPKAP kinase-2, Akt/PKB and Chk1 is rationalized bythe presence of a conserved glutamate residue at a similar location inall three kinases (Glu145 in MAPKAP kinase-2, Glu236 in Akt/PKB andGlu91 in Chk1), which in Akt/PKB forms a bidentate salt bridge with theSer−3 arginine guanidino group on GSK3-peptide. Similarly, selection fora hydrophobic residue at the Ser+1 position is explained by ahydrophobic pocket that is conserved at this position in all threekinases. The pocket is lined by Phe310, Pro314, Leu317 and Phe359 inAkt/PKB and by Met167, Leu171, Val174, Leu178 and Leu179 in Chk1. Thecorresponding Ser+1 pocket in MAPKAP kinase-2 is lined by Pro223,Pro227, Val234 and Leu235. Within this region, Gly312 in Akt/PKB andGly169 in Chk1 are replaced by Tyr225 in MAPKAP kinase-2, which mayreduce the depth of the MAPKAP kinase-2 hydrophobic pocket and explainselection for branched chain aliphatic residues in this positioncompared with Phe selection by Akt/PKB and Chk1.

The marked contrast between Arg selection at the Ser−5 position inAkt/PKB with the corresponding selection for hydrophobic residues atthis position by MAPKAP kinase-2 and Chk1 is accounted for by thepresence of Glu342 in Akt/PKB at the base of the Ser−5 pocket. Thisresidue is not conserved in MAPKAP kinase-2 and Chk1, and is insteadsubstituted by Ile255 in MAPKAP kinase-2 and by Ala200 in Chk1.Additional residues, notably Phe147, Pro189, Pro261 and Leu342 in MAPKAPkinase-2, and similarly Phe93, Ile96, Pro98, Pro133 and Leu206 in Chk1,contribute a significant hydrophobic character to this region.

MAPKAP Kinase-2 is Required for the G₂/M Checkpoint FollowingDoxorubicin Treatment.

Treatment of U2OS cells with doxorubicin generates DNA double strandbreaks, and induced a prominent G₂/M arrest between eighteen and thirtyhours following treatment (FIGS. 24A-24B). In addition to this largeG₂/M population, a minor accumulation of cells in G₁ and S phase wasalso observed. To investigate whether MAPKAP kinase-2 activation wasinvolved in the checkpoint response, RNA interference was used togenerate U2OS cells in which MAPKAP kinase-2 protein levels were stablyrepressed (FIGS. 14A-14C). Introduction of MAPKAP kinase-2-specificshRNA, but not luciferase shRNA, resulted in a robust knockdown ofMAPKAP kinase-2 protein when the entire population of transfected cellswas analyzed (FIG. 14A).

Asynchronous MAPKAP kinase-2 or luciferase shRNA knockdown cells weremock treated or exposed to doxorubicin for thirty hours, and cell cycleprogression was monitored by FACS. In one set of experiments, thespindle poison nocodazole was added to the media three hours afteraddition of doxorubicin, to cause any cells progressing through the cellcycle to arrest in mitosis. DNA content was monitored by PI staining;phospho-histone-H3 staining was used as an indicator of mitotic entry.As shown in the left panels of FIG. 14A, treatment of control luciferaseshRNA knockdown cells with doxorubicin led to the accumulation of cellswith 4N DNA content, and a lack of phospho-histone-H3 staining in eitherthe absence or presence of nocodazole. The cells expressing theluciferase shRNAs behaved identically to the untransfecteddoxorubicin-treated control U2OS cell population (FIG. 24B), with theprominent 4N DNA component and the absence of phospho-histone-H3staining indicative of an intact G₂/M checkpoint. In marked contrast,MAPKAP kinase-2-depleted cells treated with doxorubicin displayed a cellcycle profile essentially identical to that of untreated cells (FIG.14A, right upper and middle panels). Addition of nocodazole followingdoxorubicin treatment to the MAPKAP kinase-2 depleted cells caused themto accumulate in a 4N DNA containing peak, with 36.3% of the cellsstaining positively for phospho-histone H3 (FIG. 14A, right lowerpanels), a value similar to that of untreated U2OS cells blocked inmitosis with nocodazole (42%) (FIG. 24D). Identical results wereobtained using a second unrelated RNAi sequence against MAPKAP kinase-2,indicating that these results did not arise from RNAi off-targeteffects. MAPKAP kinase-2 depletion did not alter total Chk1 levels orreduce Chk1 activation following DNA damage (FIG. 14B). These findingsdemonstrate that loss of MAPKAP kinase-2 prevents cells fromestablishing a functional G₂/M checkpoint following doxorubicin-inducedDNA damage, despite the presence of activated Chk1.

MAPKAP Kinase-2 Induces Binding of Cdc25B to 14-3-3 in Response toTopoisomerase Inhibitor-Induced DNA Damage.

Two Cdc25 family members, Cdc25B and C, play important roles ininitiating and maintaining mitotic entry in normal cells, and areprominent targets of the G₂/M checkpoint. Cdc25B is believed to functionby activating Cdk1/Cyclin B at the centrosome in late G₂ as an initiatorof early mitotic events, while Cdc25C functions to further amplifyCdk1/CyclinB activity within a nuclear autoamplification loop oncemitosis has begun. In response to γ- or UV-radiation-induced DNA damage,checkpoint kinases phosphorylate Cdc25B and C on Ser323 and Ser216,respectively, to induce their binding to 14-3-3 proteins, which, alongwith a modest reduction in their catalytic activity, sequesters them inthe cytoplasm away from their nuclear cyclin/Cdk substrates. Recentstudies suggest that Cdc25B plays a particularly crucial role ininitiating and maintaining normal cell cycle G₂/M checkpoint responses,since reactivation of Cdc25B is critical for DNA-damaged cells tore-enter the cell cycle. We have shown above that MAPKAP kinase-2 iscapable of directly phosphorylating Cdc25B on Ser323 to generate the14-3-3 binding site. We therefore investigated whether MAPKAP kinase-2signaling was required for association of Cdc25B with 14-3-3 in responseto DNA damage by chemotherapeutic drugs. Control luciferase and MAPKAPkinase-2 knockdown cells were either mock treated or incubated withcisplatin, camptothecin, or doxorubicin. Cell lysates were preparedeight hours later and incubated with recombinant GST-14-3-3β/ζ. Bindingof endogenous Cdc25B to 14-3-3 was detected by immunoblotting. As shownin FIG. 14C, both doxorubicin and camptothecin treatment, but notcisplatin exposure, resulted in the generation of stable 14-3-3-bindingsites on Cdc25B in the luciferase shRNA control cells. No 14-3-3 bindingof Cdc25B, however, was detected in lysates from the MAPKAP kinase-2depleted cells (FIG. 14C, lower panel). This result is in good agreementwith the cell cycle studies in panel A, which showed loss of the G₂/Mcheckpoint in MAPKAP kinase-2 depleted cells after treatment with thetopoisomerase inhibitor doxorubicin. These data indicate that loss ofthe chemotherapy-induced G₂/M checkpoint in MAPKAP kinase-2 depletedcells likely arises, at least in part, from loss of Cdc25B binding to14-3-3 proteins.

MAPKAP Kinase-2 is Required for G₁/S Checkpoint Arrest FollowingCisplatin Treatment.

In contrast to the G₂/M checkpoint response observed indoxorubicin-treated cells, treatment with the DNA intra-strandcross-linker cisplatin caused U2OS cells to predominantly accumulate inthe G₁ and S phases of the cell cycle over the subsequent thirty hours(FIG. 24C). RNA interference was used to investigate the role of MAPKAPkinase-2 in this process. Control luciferase knockdown cells showed anidentical accumulation in G₁ and S after cisplatin exposure (FIG. 15A,left panels) as that seen in U2OS cells lacking shRNA. Addition ofnocodazole to the luciferase knockdown cells three hours followingcisplatin treatment did not reveal the appearance of any mitotic cellsover the ensuing twenty-seven hours, as monitored by phospho-histone H3staining (FIG. 15A, lower left panels), indicating a functionally intactG₁/S checkpoint. Depletion of MAPKAP kinase-2 prior to cisplatinexposure resulted in a dramatically different result. As seen in theright panels of FIG. 15A, MAPKAP kinase-2 depleted cells showed a cellcycle profile after cisplatin treatment that was similar to that ofuntreated cells other than a very slight increase in the total number ofcells in S-phase. Strikingly, when nocodazole was added three hoursfollowing cisplatin addition, the MAPKAP kinase-2 depleted cellsaccumulated in a 4N DNA containing peak with ˜42% of the cells stainingstrongly for phospho-histone H3. Identical results were obtained incells treated with a second unrelated siRNA sequence against MAPKAPkinase-2. MAPKAP kinase-2 depletion did not impair activation of Chk1after cisplatin exposure (FIG. 15B). These data imply that MAPKAPkinase-2 is essential for the cisplatin induced G₁/S arrest and thatloss of MAPKAP kinase-2 enables U2OS cells to override thecisplatin-induced G₁/S checkpoints, despite the presence of activatedChk1, and proceed into mitosis.

MAPKAP Kinase-2 is Required for Cdc25A Degradation in Response toCisplatin-Induced DNA Damage.

In contrast to the 14-3-3-mediated sequestration of Cdc25B and Cinvolved in the G₂/M checkpoint response, the G₁ and S phase checkpointsare largely controlled by the phosphorylation-dependent degradation ofanother Cdc25 isoform, Cdc25A. Based on our observation that depletionof MAPKAP kinase-2 resulted in loss of the G₁/S checkpoint response, weinvestigated whether MAPKAP kinase-2 was required for the degradation ofCdc25A following cisplatin-induced DNA damage. Luciferase shRNA controlcells and MAPKAP kinase-2 depleted cells were treated with cisplatin,and cell lysates immunoblotted for Cdc25A at eight and twelve hoursfollowing treatment (FIG. 15B). Cdc25A levels decreased dramatically inthe control luciferase knockdown cells after treatment with cisplatin.In contrast, in the MAPKAP kinase-2 depleted cells, the level of Cdc25Afollowing cisplatin exposure was only minimally reduced, and remainedcomparable to that seen in undamaged cells. These data indicate that inthe absence of MAPKAP kinase-2, U2OS cells are defective in targetingCdc25A for degradation in response to cisplatin induced DNA damage. Thisinability of MAPKAP kinase-2 depleted cells to degrade Cdc25A likelyexplains the failure of MAPKAP kinase-2 depleted cells to establish asustained G₁/S checkpoint following cisplatin exposure.

The degradation of Cdc25A in response to DNA damage involves the directphosphorylation of Cdc25A by checkpoint kinases. In response to UV andγ-irradiation, for example, Chk1 phosphorylates Cdc25A at multiple sitesfacilitating its subsequent ubiquitin-mediated destruction by theproteosome. Chk1, however, is activated normally in the MAPKAP kinase-2depleted cells after cisplatin treatment (FIG. 15B). Other kinasesbesides Chk1, such as Chk2, have been recently reported to be able tophosphorylate Cdc25A on at least some of the same sites as Chk1 undercertain conditions. Furthermore, we have shown that the optimal aminoacid sequence motif on peptides and proteins phosphorylated by MAPKAPkinase-2 is nearly identical to the optimal sequence motifphosphorylated by Chk1 and Chk2. We therefore investigated whetherCdc25A could be a direct MAPKAP kinase-2 substrate. Recombinant Cdc25Awas incubated with purified MAPKAP kinase-2 or Chk1 in vitro in thepresence of 32P-γ-ATP, and phosphorylation monitored bySDS-PAGE/autoradiography. As shown in FIG. 15C, MAPKAP kinase-2phosphorylated Cdc25A in vitro as efficiently as Chk1. Together, thesefindings suggest that degradation of Cdc25A in response to cisplatintreatment either requires direct phosphorylation of Cdc25A by MAPKAPkinase-2, or that MAPKAP kinase-2 activity is required to target Chk1 toCdc25A in vivo.

Down-Regulation of MAPKAP Kinase-2 Increases the Sensitivity of TumorCells to Chemotherapy.

The experiments in FIGS. 15A-15C and 16A-16D indicate that MAPKAPkinase-2 is critical for cisplatin- and doxorubicin-triggered G₁/S andG₂/M arrest. These checkpoint defects in MAPKAP kinase-2 depleted cellsmight render them more sensitive to the antiproliferative and cytotoxiceffects of chemotherapy. To investigate this, control or MAPKAP kinase-2knockdown U2OS cells were mock treated or incubated with increasingdoses of cisplatin or doxorubicin for eight hours, washed, trypsinizedand replated, and assayed for colony formation eight days later (FIGS.16A-16B). When compared to the control shRNA-treated cells, MAPKAPkinase-2 depleted cells displayed a dramatically increased sensitivityto both cisplatin and doxorubicin treatment, particularly at relativelylow drug doses (FIGS. 16C-16D). For example, luciferase shRNA cellstreated with either 10 μM cisplatin or 1 μM doxorubicin formed ˜40% ofthe number of colonies as those formed by untreated cells, while inMAPKAP kinase-2-depleted cells, these same cisplatin and doxorubicintreatments reduced the number of colonies to only 4% and 2%,respectively, of those seen in the untreated cells.

To establish whether the absence of MAPKAP kinase-2 could also enhancethe anti-tumorigenic effect of cisplatin or doxorubicin in vivo, weintroduced control or MAPKAP kinase-2 siRNAs into H-Ras-V12 transformedp53^(−/−) MEFs, treated them with either vehicle alone, 1 μM cisplatinor 0.1 μM doxorubicin, and then implanted them into nude mice. Eachanimal received two injections of MAPKAP kinase-2 siRNA-transfectedcells (left flank), and two injections of control siRNA transfectedcells (right flank), and tumor formation was assessed at fifteen days.FIG. 17A, left panel, shows that in the absence of treatment with DNAdamaging agents, all four injections resulted in formation of solidfibrous tumors after fifteen days. In general, the size of the tumorsresulting from injection of MAPKAP kinase-2 depleted cells was largerthan that from control siRNA-transfected cells (FIGS. 17A, 17B, and17D). Pre-treatment of the control siRNA transfected cells with eithercisplatin or doxorubicin prior to implantation did not prevent tumorformation. The resulting tumors, however, were reduced to ˜35% of thesize and weight of the tumors formed by untreated cells (FIGS. 17A and17D). Depletion of MAPKAP kinase-2 prior to treatment with eithercisplatin or doxorubicin completely eliminated the formation of tumors(FIGS. 17A, 17C, and 17D), indicating that the enhanced sensitivity ofthese cells to chemotherapeutic drugs seen in culture was maintainedeven when the cells were grown within a normal tissue microenvironment.

Taken together with the loss of G₁/S and G₂/M checkpoints observed byFACS analysis, and the mis-regulation of the mitotic phosphatases Cdc25Aand B (FIGS. 14A-14C and 15A-15C), these data provide strong evidencethat down-regulation of MAPKAP kinase-2 activity results in enhancedsensitivity of cells to genotoxic stress in vitro and in vivo. Thesefindings have potential therapeutic implications, since they suggestthat targeting of MAPKAP kinase-2 with small molecule inhibitors shouldresult in an enhanced sensitivity of tumor cells to conventionalchemotherapeutic agents.

MAPKAP Kinase-2 and Chk1 are Activated Independently.

The activation of MAPKAP kinase-2 by cisplatin, camptothecin,doxorubicin, and UV irradiation that we observed is strikingly similarto the activation profile reported for Chk1. Similarly, the impairedG₁/S and G₂/M checkpoints seen after these DNA damaging stimuli inMAPKAP kinase-2 knockdown cells bears some resemblance to what has beenreported for Chk1-deficient cells. These phenotypic similaritiesprompted us to further investigate whether the activation of Chk1 andMAPKAP kinase-2 was interdependent. As shown in FIGS. 14B and 15B,activation of Chk1 in response to cisplatin and doxorubicin wasunimpaired in MAPKAP kinase-2 depleted cells. We therefore investigatedthe opposite possibility—whether the activation of MAPKAP kinase-2 afterDNA damage was dependent on Chk1. U2OS cells were depleted of Chk1 usingsiRNA, exposed to cisplatin and doxorubicin, and analyzed for activationof MAPKAP kinase-2. As shown in FIG. 19, phosphorylation/activation ofMAPKAP kinase-2 occurred normally after treatment with these DNAdamaging agents, regardless of the presence or absence of Chk1. Thus,activation of MAPKAP kinase-2 and Chk1 after genotoxic stress appears tooccur independently of each other.

The MAPKAP Kinase-2 DNA Damage Checkpoint Phenotype can be SyntheticallyRescued by Chk1 Overexpression.

The observation that Chk1 and MAPKAP kinase-2 phosphorylate the sameoptimal sequence motif, target a set of overlapping substrates, and areactivated independently of each other, prompted us to perform a geneticexperiment to investigate whether loss of MAPKAP kinase-2 could berescued by overexpression of Chk1 in mammalian cells (FIGS. 20A-20E). Inthese experiments, luciferase- or MAPKAP kinase-2 shRNA-expressing cellswere transiently transfected with a mammalian Chk1 expression construct,or with an empty vector control (FIG. 25). Cells were exposed tocisplatin, doxorubicin, or UV radiation thirty hours followingtransfection, harvested after an additional thirty hours, and cell cycleprogression analyzed by FACS. In one set of experiments, nocodazole wasadded to the media three hours following addition of chemotherapy or UV,to cause any cells progressing through the cell cycle to arrest inmitosis.

Consistent with what we observed previously, luciferase shRNA controlcells transfected with the empty vector DNA executed a G₁/S arrestfollowing exposure to cisplatin (FIG. 20A) and UV irradiation (FIG. 26),and displayed a prominent G₂ arrest in response to doxorubicin (FIG.20B). These cell cycle profiles were unchanged when the luciferase shRNAcells were transfected with Chk1. MAPKAP kinase-2 depleted cellstransfected with empty vector DNA broke through both checkpoints andaccumulated in mitosis when nocodazole was added to the media (FIGS.20A-20B). Overexpression of Chk1 in the MAPKAP kinase-2 depleted cells,however, completely restored their ability to establish functionalcheckpoints following genotoxic stress. The cells now arrested in G₁/Sin response to cisplatin and UV irradiation, and in G₂ followingdoxorubicin (rightmost panels in FIGS. 20A and 20B, and FIG. 26).Addition of nocodazole to the growth media of these MAPKAP kinase-2depleted Chk1 over-expressing cells did not increase the number ofphosphohistone H3 positive cells. Thus, overexpression of Chk1 preventedMAPKAP kinase-2 depleted cells from progressing through the cell cycleafter genotoxic stress.

We investigated whether the synthetic rescue of G₁/S and G₂/Mcheckpoints by Chk1 in MAPKAP kinase-2 depleted cells was alsosufficient to reduce their susceptibility to chemotherapeutic treatment.Luciferase and MAPKAP kinase-2 knockdown cells transfected with Chk1 orvector alone were mock treated or incubated with increasing doses ofcisplatin and doxorubicin for eight hours, or irradiated with 20 J/m² ofUV light. Cells were washed, trypsinized, replated and assayed forcolony formation after eight days as described previously (FIGS. 27-28).As summarized in FIGS. 20C-20E, MAPKAP kinase-2 depleted cells,transfected with the empty control vector, showed enhanced sensitivityto the anti-proliferative effects of cisplatin, doxorubicin and UV.Overexpression of Chk1 in these MAPKAP kinase-2 depleted cells restoredtheir clonogenic survival to levels that were indistinguishable fromthose seen with control cells containing wild-type levels of MAPKAPkinase-2.

UCN-01 is a Potent Inhibitor of both Chk1 and MAPKAP Kinase-2.

The staurosporine derivative 7-hydroxystaurosporin/UCN-01 inhibits Chk1with an IC₅₀ that is ˜1000 fold lower than that for Chk2, and hence hasbeen used experimentally as a Chk1-specific inhibitor. Strongcircumstantial evidence, however, suggests that UCN-01 inhibits otherkinases involved in cell cycle control at similar concentrations asthose used for Chk1 inhibition studies. For example, Chk1-depleted cellsmaintain phosphorylation of Cdc25C on Ser−216 both during asynchronousgrowth and following γ-irradiation. Phosphorylation at this site is lostwhen cells are treated with low doses of UCN-01 (˜300 nM), indicatingthat UCN-01 inhibitable kinase(s) other than Chk1 participate in Cdc25Cphosphorylation. Based on our finding that MAPKAP kinase-2 is a criticalcheckpoint regulator, we investigated whether UCN-01 inhibited MAPKAPkinase-2 at doses typically used in Chk1 inhibition experiments. Invitro kinase assays were performed with Chk1 and MAPKAP kinase-2 usingan optimal peptide substrate with the core consensus sequence LQRQLSI(SEQ ID NO: 16), similar to the 14-3-3 binding sequence in Cdc25B and C,in the presence of various concentrations of UCN-01. As shown in FIG.21A, UCN-01 potently inhibited both kinases, with an IC₅₀ value of 35 nMfor Chk1 and ˜95 nM for MAPKAP kinase-2. The IC₅₀ value we measured forChk1 is in good agreement with previously published data. Importantly,the IC₅₀ value we measured for MAPKAP kinase-2 is significantly belowthe concentrations of UCN-01 that are used in “Chk1-specific” checkpointabrogation assays, suggesting that under the conditions used in thosestudies, both Chk1 and MAPKAP kinase-2 were being inhibited.

To examine the structural basis for UCN-01 inhibition of MAPKAPkinase-2, the structure of the MAPKAP kinase-2:UCN-01 complex wasmodeled using coordinates from the published MAPKAPkinase-2:staurosporine structure, and compared the results with theco-crystal structure of Chk1:UCN-01 (FIG. 21B). As seen in panels 2, 3and 5 of FIG. 21B, the 7-hydroxy moiety of UCN-01 can be easilyaccommodated into the MAPKAP kinase-2:staurosporine structure, where itsclosest neighboring residues would be Vail 18 (2.8 Å to Cγ2), Leu141(3.2 Å to Cδ1), and Thr206 (3.6 Å to Cγ2). This lack of sterichindrance, and the overall similarity of the modeled MAPKAPkinase-2:UCN-01 structure to the Chk1:UCN-01 structure (panels 1 and 4of FIG. 21B), provides a structural rationale for the tight bindingobserved biochemically.

To verify that MAPKAP kinase-2 is a direct target of UCN-01 in cells, wemeasured the phosphorylation of the MAPKAP kinase-2-specific substratehsp-27 after heat shock, a stimulus that activates the p38 MAPK/MAPKAPkinase-2 pathway. Control luciferase shRNA expressing or MAPKAP kinase-2shRNA expressing U2OS cells were incubated at 42° C. or 37° C. for twohours in the presence or absence of 250 nM UCN-01, and phosphorylationof hsp-27 monitored by immunoblotting with an antibody against pSer82, awell established MAPKAP kinase-2 phosphorylation site. FIG. 21C showsphosphorylation of hsp-27 when the control luciferase shRNA cells wereplaced at 42° C. (lane 1). This phosphorylation was completely abrogatedby treatment with UCN-01 (lane 2). No phosphorylation was observed inMAPKAP kinase-2 knockdown cells placed at 42° C. regardless of thepresence or absence of UCN-01 (lanes 3, 4). Likewise, no signal wasobserved in both the control and MAPKAP kinase-2 knockdown cells thatwere maintained at 37° C., with or without UCN-01 treatment (lane 5-8).Furthermore, heat shock was equally effective in promoting thephosphorylation of hsp-27 on Ser−82, and UCN-01 was equally effective inblocking Ser−82 phosphorylation in cells that were depleted of Chk1(FIG. 21D, lanes 1-4). Thus, UCN-01 inhibition of MAPKAP kinase-2 invivo is independent of Chk1 function. These findings provide strongevidence that UCN-01 is a direct inhibitor of MAPKAP kinase-2 withincells, and suggest that the clinical efficacy of UCN-01 in cancertreatment likely arises from the simultaneous inhibition of two parallelbut non-redundant checkpoint pathways involving Chk1 and MAPKAPkinase-2.

Since disruption of the MAPKAP kinase-2 signaling pathway enhanceschemotherapeutic responses even in the presence of a functional Chk1response, and since MAPKAP kinase-2 knock-out mice are viable, incontrast to Chk1 knock-out mice, our results suggest that a MAPKAPkinase-2 specific inhibitor might provide significant clinical benefitwith fewer undesirable side-effects. In either case, our current datastrongly support the development of clinical MAPKAP kinase-2 inhibitorsas viable anti-cancer agents. Given the dependence of p53-defectivecells on intra-S and G₂/M checkpoint pathways, targeting MAPKAP kinase-2may be a particularly efficacious approach to treating these types ofhuman cancers. Thus, therapeutic treatments that interfere with MAPKAPkinase-2 function would be expected to preferentially sensitizep53-deficient cells to treatment with DNA-damaging chemotherapeuticdrugs without similarly sensitizing wild-type cells. Disorders, e.g.,neoplastic disorders, that include p53-deficient cells could be treatedeffectively and specifically using therapy that combines administrationof a MAPKAP kinase-2-interfering compound, e.g., UCN-01, and one or morechemotherapeutic agents, preferably at substantially lower levels thanwould otherwise be necessary to treat the disorder, thereby largelysparing normal cells from the deleterious effects of chemotherapy.

Model for the Role of MAPKAP Kinase-2

Our data show that a crucial role of p38 SAPK in response to UV-inducedDNA damage is the phosphorylation and activation of MAPKAP kinase-2,leading to MAPKAP kinase-2-directed phosphorylation of Cdc25 familymembers to induce 14-3-3-binding and subsequent cell cycle arrest. Inthis way, MAPKAP kinase-2 performs similar functions after UV-C inducedDNA damage as those performed by Chk1 and Chk2 after exposure of cellsto ionizing radiation.

MAPKAP kinase-2 undergoes initial activation in the nucleus withsubsequent export of the active kinase to the cytoplasm. Thus, MAPKAPkinase-2 is well-positioned to function as both a nuclear initiator ofCdc25B/C phosphorylation in response to DNA damage, and as a maintenancekinase that keeps Cdc25B/C inhibited in the cytoplasm. A unified modelfor kinase-dependent DNA damage checkpoints is presented in FIG. 7. Inresponse to ionizing radiation, ATM activation of Chk2 and ATRactivation of Chk1 leads to phosphorylation of Cdc25 family members onrelated sequences corresponding to the checkpoint kinase core “motif”LXRXX[S/T][Hydrophobic] (SEQ ID NO: 18). Similarly, in response toUV-induced DNA damage, ATR activates Chk1 and p38 SAPK activates MAPKAPkinase-2, leading to phosphorylation of the same core motif on Cdc25family members. The major role of Chk1 appears to involvephosphorylation of Cdc25A after IR, whereas Chk2 appears tophosphorylate all three Cdc25 family members. In the absence of Chk2,Chk1 appears to be able to subsume at least part of this function. Ourdata now indicate that MAPKAP kinase-2 is the primary effector kinasethat targets Cdc25B/C after Uv-C exposure. MAPKAP kinase-2 may also beinvolved in Cdc25A phosphorylation, since we observed that the G₁ andS-phase checkpoints were eliminated in the MAPKAP kinase-2 knockdowncells.

The results presented here indicate that the activities of both Chk1 andMAPKAP kinase-2 are required for G₁/S and G₂/M cell cycle arrest inresponse to DNA damaging chemotherapy and UV-irradiation (FIG. 22). At asystems level, these observations suggest that the normal DNA damagecheckpoint response involves the unified actions of a dedicated DNAdamage response pathway (i.e., Chk1) and a potentially more globalstress response pathway (MAPKAP kinase-2). Individual kinase activitiesemerging from each of these pathways appear to be titered to levelsthat, in combination, are just adequate to arrest the cell cycle afterdamage, presumably facilitating rapid checkpoint release once the DNAdamage has been repaired. In agreement with this hypothesis,overexpression of Chk1 rescued both the G₂/M and G₁/S cell cyclecheckpoint defects observed in MAPKAP kinase-2 depleted cells.

Experimental Procedures

Chemicals, antibodies, and drugs. UCN-01 was the kind gift of R.Schultz, Drug Synthesis and Chemistry Branch, Developmental TherapeuticsProgram, Division of Cancer Treatment and Diagnosis, National CancerInstitute (Bethesda, Md.). Cisplatin, doxorubicin and camptothecin,puromycin, and glutathione beads were purchased from Sigma-Aldrich.Propidium iodide was purchased from Calbiochem. Antibodies against totaland phosphorylated forms of MAPKAP kinase-2, p38 MAPK, Chk1, Chk2,ATM/ATR substrate, hsp-27, and p53 (pS20) were purchased from CellSignaling Technology (Beverly, Mass.). Antibodies against β-actin and5-bromo-2-deoxyuridine (BrdU) were purchased from Sigma-Aldrich; ananti-Cdc25A antibody (MS-640-P1, cocktail) was from NeoMarker (Fremont,Calif.); an anti-Cdc25B antibody was from Transduction Labs, an anti-GSTantibody was from Amersham/GE Healthcare, and an anti-phospho histone H3antibody was from Upstate. Active MAPKAP kinase-2 was purchased fromUpstate. Propidium Iodide (PI) was purchased from Calbiochem, amylosebeads were purchased from New England Biolabs, Ni-NTA agarose werepurchased from QIAGEN, and glutathione beads and BrdU were purchasedfrom Sigma-Aldrich.

Cell culture. U2OS cells, HeLa cells, U87MG cells and H-Ras-V12transformed p53^(−/−) MEFs were cultured in DMEM supplemented with 10%FCS and penicillin/streptomycin at 37° C. in a humidified incubatorsupplied with 5% CO2. GM05849 A-T fibroblasts and the correspondingcontrol GM00637 fibroblasts, and GM18366 ATR-defective Seckel syndromefibroblasts and the corresponding control GM00023 fibroblasts wereobtained from the Coriell cell repository and were cultured in MEMsupplemented with Eagle's salts, 10% FCS, and penicillin/streptomycin.

Purification of recombinant proteins. Constructs encoding GST- andMBP-fusion proteins were transformed into DH5α or BL21(DE3) strains ofE. coli and recombinant proteins obtained by inducing late log-phasecells with 0.4 mM IPTG at 37° C. for three to five hours. Cells werelysed by sonication in lysis buffer containing 50 mM Tris-HCl, pH 7.5,250 mM NaCl, 1 mM DTT, 8 μg/mL pepstatin, 8 μg/mL aprotinin, and 8 μg/mLleupeptin. Fusion proteins were purified from cell lysates by usingamylose or glutathione beads. Following extensive washing with PBScontaining 0.5% NP-40 and a final wash with PBS, fusion proteins wereeluted from the beads with HEPES, pH 7.2, containing 40 mM maltose or 20mM glutathione, followed by exchange into PBS using duplicate SephadexG-25 columns (NAP-10 columns, Pharmacia). Protein concentrations weredetermined using the bicinchoninic acid assay (Pierce) as recommended bythe manufacturer, using BSA as the standard. Full-length Chk1-GST orfull-length Chk2-His6 in pFASTBAC was expressed in Sf9 insect cells.Chk1-expressing cells were lysed in buffer containing 50 mM Tris-HCl, pH7.5, 250 mM NaCl, 1 mM DTT, 1.0% NP-40, 8 μg/μL pepstatin, 8 μg/mLaprotinin, 8 μg/mL leupeptin, 2 mM Na₃VO4, 10 mM NaF, and 1 μMmicrocystin, and Chk1 was purified using glutathione beads. Chk1 waseluted from the beads with 10 mM glutathione in 50 mM Tris-HCl, pH 8.0,and dialyzed into kinase buffer. Chk2 expressing cells were lysed inbuffer containing 50 mM Tris-HCl, pH 7.5, 250 mM NaCl, 1 mM DTT, 1.0%NP-40, 8 μg/mL pepstatin, 8 μg/mL aprotinin, 8 μg/mL leupeptin, 2 mMNa₃VO₄, 10 mM NaF, and 1 μM microcystin, and Chk2 was purified usingNi-NTA agrose beads. After washing extensively with lysis buffercontaining 40 mM imidazole, Chk2 was eluted from the beads with 100 mMimidazole in 50 mM Tris-HCl, pH 8.0, and dialyzed into kinase buffer.

Point mutations were generated using the Stratagene Quick ChangeMutagenesis Kit and confirmed by sequencing the entire coding regions.

Kinase motifscreening with oriented peptide libraries and in vitrokinase assays. Using the methods of the invention, one skilled in theart would be able to utilize a peptide library screen to identifypeptides that bind to a p38 SAPK polypeptide, MAPKAP kinase-2polypeptide, or other biologically relevant target. Peptides identifiedin such a screen, or related compounds, would have potential therapeuticbenefit due to their ability to inhibit the biological activity of,e.g., a MAPKAP kinase-2 polypeptide.

Combinatorial peptide library screening was performed using recombinantpurified p38α SAPK, MK2, Chk1 and Chk2 as previously described (Songyangand Cantley, Methods Mol. Biol., 87:87-98, 1998) with minormodifications. Briefly, 5.0 μg of recombinant p38α SAPK, 3.0 μg MK2, 2.0μg Chk1 and 2.0 μg Chk2 were incubated with 1 mg of each peptide libraryin 300 μl reaction volumes containing 20 mM HEPES, pH 7.5, 10 mM MgCl₂,3 mM 2-mercaptoethanol, and 100 μM ATP containing 2.5 μCi of 32P-γ-ATPfor 120 min at 30° C. Under these conditions, approximately 1% of thepeptide mixture was phosphorylated. The reaction mixture was diluted byaddition of 300 μl of 30% acetic acid, and the phosphorylated peptidesseparated from unincorporated 32P-γ-ATP by DEAE column chromatography (1ml bed volume) using isocratic elution with 30% acetic acid. The peptidemixture (both phosphorylated and unphosphorylated, but free of ATP)eluted within the first 1 ml following the 600 μl void volume of thecolumn. Samples were dried in a Speed-Vac apparatus.

For the p38α SAPK peptide library experiments, the sample wasresuspended in 200 μl of 50 mM MES, pH 5.5, containing 1 M NaCl.Separation of phosphorylated from non-phosphorylated peptides wasachieved by IMAC using ferric-iminodiacetic acid beads. A 0.5 mliminodiacetic acid column was charged with 2.5 ml of 20 mM FeCl₃ andextensively washed with H₂O, then with 3 ml of 500 mM NH₄HCO₃, pH 8.0, 3ml of H₂O, and 3 ml of 50 mM MES (pH 5.5)/1 M NaCl. The peptide mixturewas applied and the column was developed with 3 ml 50 mM MES, pH 5.5, 1M NaCl, followed by 4 ml of H₂O to remove nonphosphorylated peptides.Phosphorylated peptides were then eluted with 2 ml of 500 mM NH₄HCO₃, pH8.0, and dried in a Speed-Vac apparatus, and resuspended in 80 μl H2O.

Peptide library screens using basophilic kinase-directed libraries arecomplicated by a high background of non-phosphorylated Asp/Glu-richpeptides that co-purified with the phosphorylated peptides during theimmobilized metal affinity chromatography (IMAC) step prior to peptidesequencing, greatly complicating the analysis. To overcome this problem,we developed a new approach in which peptide libraries are firstphosphorylated by the kinase of interest, and then treated withmethanolic HCl to convert Asp and Glu residues to their uncharged methylesters. Using this approach, the background of nonphosphorylatedpeptides that adhere to the IMAC column was reduced to insignificantlevels. Furthermore, the Asp and Glu methyl esters were converted backto their free acids during the sequencing reaction, allowing selectionfor these residues, if present in the phosphorylation motif, to beaccurately measured.

For the MAPKAP kinase-2, Chk1, and Chk2 peptide library experiments, 40μl of thionyl chloride was added dropwise in a hood to 1 ml of drymethanol. This solution was then used to dissolve each of the driedpeptide libraries, followed by stirring at room temperature for onehour. The peptide library was dried down overnight and resuspended in100 μl of a 1:1:1 mixture of methanol/acetonitrile/water. A 0.5 mliminodiacetic acid column was charged with 2.5 ml of 20 mM FeCl₃ andextensively washed with H₂O, then with 3 ml of 500 mM NH₄HCO₃ (pH 8.0),3 ml of H₂O, and 3 ml of 50 mM MES, pH 5.5, 1 M NaCl. The peptidemixture was applied and the column was developed with 4 ml of H₂Ofollowed by 3 ml NH₄HCO₃, pH 8.0, to remove non-phosphorylated peptides.Phosphorylated peptides were eluted with 2 ml of 500 mM NH₄HCO₃, pH11.0, dried in a Speed-Vac apparatus, and resuspended in 40-80 μl H₂O.

Following IMAC purification, libraries (0.5-1.5 nmoles) were subjectedto automated Edman sequencing using an Applied Biosystems model 477Apeptide sequencer. Data analysis was performed by normalizing theabundance (mol-%) of each amino acid in the phosphorylated peptidemixture to that present in the starting libraries. The sums of the finalpreference ratios were normalized to the total number of amino acids inthe degenerate positions within the peptide libraries so that aparticular amino acid would have a preference value of 11n the absenceof selectivity at a particular position. The degenerate peptidelibraries used for in vitro kinase screening with p38 MAP kinase, MK2,Chk1, and Chk2 consisted of the sequences GAXXXXSPXXXXAKKK [SP library](SEQ ID NO: 19), where X denotes all amino acids except Cys, Ser, Thr,and Tyr; GAXXXXPXSPXXXXXAKKK [PxSP library] (SEQ ID NO: 20), where Xdenotes all amino acids except Cys; or GAXXSXXXXAKKK [RxxS library] (SEQID NO: 21), where X denotes all amino acids except Cys, Ser, Thr andTyr. In all libraries, S denotes Ser, P denotes Pro, and R denotes Arg.

Kinase reactions were performed in 30 μl of kinase reaction buffer (20mM HEPES, pH 7.5, 10 mM MgCl₂, 3 mM 2-mercaptoethanol, 100 μg/ml BSA, 50μM ATP, 10 μCi 32P-γ-ATP) containing 2.0 μg of recombinant p47 or Cdc25Bsubstrate protein or the specified amount of peptide and 0.10 μg ofrecombinant p38a SAPK or 0.03 μg of recombinant MAPKAP kinase-2 at 30°C. for the indicated time. The sequences of the p38 optimal peptide andthe p47phox peptide were KKAZGPQGPQSPIE (SEQ ID NO: 22) andKKAZGPQSPGSPLE (SEQ ID NO: 23), respectively. For 14-3-3 pulldowns ofCdc25B following in vitro phosphorylation by p38 or MAPKAP kinase-2, 2.0μg of Cdc25B was incubated with 10-fold excess 14-3-3-MBP and analyzedby autoradiography. For kinetic measurements, the reactions wereterminated by the addition of an equal volume of 0.5 percent phosphoricacid, and 5 μl was spotted onto p81 paper. The p81 paper was washed 5×in 0.5 percent phosphoric acid and added to scintillation fluid forscintillation counting. For in vitro phosphorylation reactions, thereactions were terminated by the addition of an equal volume of samplebuffer followed by heating at 95° C. for 3 min. Samples were analyzed bySDS-PAGE followed by transfer to nitrocellulose for autoradiography andimmunoblotting. The rate of p38α phosphorylation of isolated peptidesand full-length p47phox proteins was determined by scintillationcounting using peptide concentrations of 100, 400, and 1400 μM, andprotein concentrations of 1, 5, 10 and 15 μM, with time points taken atfive, ten, and twenty minutes. MAPKAP kinase-2 phosphorylation ofMK2tides was performed using peptide concentrations of 5, 10, 20, 40,80, 160, 320, 500, and 1000 μM, with time points taken at three, six,nine, and twelve minutes. From these enzymatic studies, K_(m), V_(max)and V_(max)/K_(m) values were then ascertained. All kinetic experimentswere performed a minimum of three times. For each experimental conditionin the determination of the K_(m) and V_(max) values, we verified thatthe reaction rates were linear with respect to time for all substrateconcentrations and that less than 10% substrate was phosphorylated.

In vitro kinase assays for UCN-01 IC₅₀ determination were performed in30 μl reactions containing 20 mM HEPES (pH 7.5), 10 mM MgCl2, 3 mM2-mercaptoethanol, 100 μg/ml BSA, 50 mM ATP, 10 μCi 32P-γ-ATP, and 50μM. MK2-tide substrate for twenty minutes at 30° C. Chk1 was used at aconcentration of 0.3 μM; MAPKAP kinase-2 was used at a concentration of0.1 μM. Reactions were terminated by adding an equal volume of 0.5%phosphoric acid to the reaction and 5 μl was spotted onto P81 paper.After washing 5× in 0.5% phosphoric acid, sample were subjected toscintillation counting. Cdc25A phosphorylation studies were performedusing GST-Cdc25A immunoprecipitated from HEK293T cells transfected withpCMV GST-Cdc25A, a generous gift from Dr. W. Harper (Harvard MedicalSchool). In brief, HEK293T cells were transfected with pCMV GST-Cdc25Aconstruct using the calcium phosphate method described earlier. Cellswere harvested thirty-six hours later, lysed in a buffer containing 50mM Tris-HCl, pH 7.8, 150 mM NaCl, 1.0% NP-40, 5 mM EDTA, 2 mM DTT, 8μg/ml pepstatin, 8 μg/ml aprotinin, 8 μg/ml leupeptin, 2 mM Na₃VO4, 10mM NaF, and 1 μM microcystin and cleared by centrifugation. Supernatantswere precleared with protein G beads for one hour. GST-Cdc25A wasprecipitated with 50 μl GSH beads (Sigma-Aldrich). Beads were washedfive times in kinase buffer and used in kinase reactions. Kinasereactions were performed in 50 μl of kinase reaction buffer using 0.3 μMChk1 and 0.1 μM MAPKAP kinase-2. Reactions were performed at 30° C. fortwenty minutes and terminated by addition of 50 μl 2× sample buffer.Samples were heated at 95° C. for three minutes, separated on a 12.5%SDS-PAGE, and visualized using a phosphor imager (Molecular Dynamics).

14-3-3 pull-down assays, immunoblotting, and immunofluorescence. U2OScells were lysed in lysis buffer: 50 mM Tris/HCl, pH7.8, 150 mM NaCl,1.0% NP-40, 5 mM EDTA, 2 mM DTT, 8 μg/ml pepstatin, 8 μg/ml aprotinin, 8μg/ml leupeptin, 2 mM Na₃VO₄, 10 mM NaF, 1 μM microcystin for twentyminutes at 4° C. Clarified lysates (0.5-2 mg protein) were incubatedwith 20 μL glutathione beads or amylose beads containing 10-20 μg14-3-3-GST or 14-3-3-MBP, respectively, for 120 minutes at 4° C.Following washing, lysates and bead-bound proteins were analysed bySDS-PAGE, followed by transfer to PVDF membranes and immunoblotted withthe indicated antibodies. For immunofluorescence experiments, U2OS cellswere seeded onto 18 mm² coverslips, irradiated or mock-treated, fixed,extracted, and immunostained as described previously (Clapperton et al.,Nat. Struct. Mol. Biol., 11:512-518, 2004).

FACS analysis. UV irradiation was performed at 254 nm (UV-C) using aStratalinker 2400 (Stratagene). U2OS cells were fixed in 70% ethanolovernight at −20° C., permeabilized with PBS containing 0.2% TritonX-100 for twenty minutes at 4° C., blocked with 2% FBS in PBS, andincubated with 1 μg of anti-phospho-histone H3 per 10⁶ cells for sixtyminutes on ice. Following washing, cells were incubated withFITC-conjugated goat anti-rabbit antibody (diluted 1:500) for thirtyminutes on ice, washed, and resuspended in PBS containing 50 μg/ml PIfor twenty minutes immediately prior to FACS analysis. Analysis wasperformed using a Becton Dickinson FACS machine with CellQuest software.

For BrdU incorporation experiments, cells were incubated with 30 μM BrdUfor the indicated times, then fixed and permeabilized as above. Cellswere denatured in 2N HCl for twenty minutes at room temperature,neutralized with 0.1M Na₂B₄O₇ (pH 8.5), blocked with 2% FBS in PBS, andincubated with a murine anti-BrdU antibody for sixty minutes on ice.Following washing, cells were incubated with FITC-conjugated goatanti-mouse antibodies and PI as above. Analysis was performed using aBecton Dickinson FACS machine with CellQuest software.

Clonogenic survival assay. Cells were either mock-treated or treatedwith increasing doses of doxorubicin or cisplatin. After eight hours oftreatment, cells were washed three times with growth media and threetimes with PBS, trypsinized and replated at a concentration of 5000cells/10 cm² dish. After eight days, cells were fixed and stained with0.1% crystal violet (Sigma-Aldrich). Colonies consisting of >50 cellswere counted, and surviving fractions were determined by normalizationagainst untreated cells. Experiments were performed in triplicate andare plotted as mean values with standard deviations indicated by theerror bars.

Murine tumor models. H-Ras-V 12-transformed p53^(−/−) MEFs were used forin vivo tumor formation assays. Cells were transfected with siRNAoligonucleotides against GFP or murine MAPKAP kinase-2 for forty-eighthours, then mock treated or incubated with 0.1 μM doxorubicin or 1 μMcisplatin for eight hours, washed three times in growth media, threetimes in PBS, trypsinized, and resuspended at a concentration of 10⁷cells/ml in PBS. 10⁶ cells were subcutaneously injected into the flanksof nude mice (Ncr nu/nu, Taconic).

For tumor regression assays, H-Ras-V12 transformed p53^(−/−) MEFs werestably transfected with a lentiviral transfer vector encoding for shRNAtargeting either MAPKAP kinase-2 or luciferase. 10⁶ cells were injectedinto the flanks of nude mice as above, and tumors were allowed to formfor twelve days. Mice were then treated with either cisplatin (2 mg/kg,intraperitoneal administration 3× per week) or doxorubicin (4 mg/kg,intraperitoneal administration 3× per week), monitored for a total oftwenty-six days, and then sacrificed. Tumor diameter was measuredperiodically during growth and tumors were weighed at the endpoint.Experiments were performed in quadruplicate, and data plotted as samplemeans with error bars showing standard deviation.

Structural modeling. Activated MAPKAP kinase-2 (phosphorylated on Thr222) was modeled using the crystal structure of the ADP complex(Underwood et al., Structure, 11:627-636, 2003) with the activation loop(residues 213 to 241) deleted and rebuilt using the corresponding region(residues 299 to 328) from the structure of activated Akt/PKB in complexwith AMP-PNP and GSK3-peptide (Yang et al., Nat. Struct. Biol.,9:940-944, 2002) as a template. An optimal peptide, LQRQLSIA (SEQ ID NO:6), was modeled in the active site based on the GSK3-peptide.Coordinates for the activated MAPKAP kinase-2/peptide complex are listedin Table 1 in standard Protein Data Bank (PDB) format (details about theProtein Data Bank and the associated format for coordinates may be foundin Berman et al., Nuc. Acids Res., 28:235-242, 2000). Table 2 listspairs of atoms in the complex that form the closest protein-peptidecontacts and that are useful for designing or identifying additionalmolecules that bind in the active site. A substrate peptide, LYRSPSMPL(residues 211-219 of human Cdc25C) (SEQ ID NO: 7) in the Chk1 activesite was similarly modeled using the GSK3-peptide as a template andmanually adjusted to resemble the published model (Chen et al., Cell,100:681-692, 2000). Structures were superimposed using ALIGN andSUPERIMPOSE. Manual adjustments of the models were made using XFIT fromthe XtalView suite.

The structure of MAPKAP kinase-2 bound to UCN-01 was modeled using PyMOLwith the structure of MAPKAP kinase-2 bound to staurosporine (PDB ID1NXK) as a base model.

TABLE 1 Coordinates of the activated MAPKAP kinase-2/peptide complexATOM 1 N PHE A 46 214.820 109.707 179.069 1.00 118.35 N ATOM 2 CA PHE A46 214.336 108.388 178.678 1.00 109.39 C ATOM 3 C PHE A 46 215.483107.382 178.556 1.00 92.52 C ATOM 4 O PHE A 46 216.008 107.116 177.4831.00 93.11 O ATOM 5 CB PHE A 46 213.617 108.523 177.335 1.00 106.31 CATOM 6 CG PHE A 46 212.712 107.345 177.122 1.00 100.53 C ATOM 7 CD1 PHEA 46 213.246 106.138 176.688 1.00 94.28 C ATOM 8 CD2 PHE A 46 211.347107.468 177.345 1.00 81.06 C ATOM 9 CE1 PHE A 46 212.408 105.049 176.4801.00 71.80 C ATOM 10 CE2 PHE A 46 210.515 106.370 177.135 1.00 83.85 CATOM 11 CZ PHE A 46 211.041 105.159 176.704 1.00 54.26 C ATOM 12 N HIS A47 215.896 106.847 179.719 1.00 99.95 N ATOM 13 CA HIS A 47 216.976105.867 179.715 1.00 110.68 C ATOM 14 C HIS A 47 216.467 104.473 179.3421.00 99.74 C ATOM 15 O HIS A 47 215.591 103.903 179.979 1.00 108.34 OATOM 16 CB HIS A 47 217.598 105.835 181.111 1.00 128.34 C ATOM 17 CG HISA 47 217.973 107.234 181.527 1.00 172.75 C ATOM 18 ND1 HIS A 47 219.157107.813 181.211 1.00 209.78 N ATOM 19 CD2 HIS A 47 217.216 108.147182.270 1.00 195.55 C ATOM 20 CE1 HIS A 47 219.113 109.047 181.749 1.00214.52 C ATOM 21 NE2 HIS A 47 217.963 109.275 182.389 1.00 216.85 N ATOM22 N VAL A 48 217.023 103.942 178.238 1.00 93.29 N ATOM 23 CA VAL A 48216.620 102.613 177.798 1.00 69.76 C ATOM 24 C VAL A 48 217.795 101.841177.194 1.00 72.39 C ATOM 25 O VAL A 48 218.595 102.370 176.434 1.0067.87 O ATOM 26 CB VAL A 48 215.510 102.770 176.757 1.00 60.38 C ATOM 27CG1 VAL A 48 215.345 101.470 175.973 1.00 76.52 C ATOM 28 CG2 VAL A 48214.200 103.110 177.443 1.00 87.98 C ATOM 29 N LYS A 49 217.962 100.570177.519 1.00 63.04 N ATOM 30 CA LYS A 49 219.047 99.811 176.921 1.0061.31 C ATOM 31 C LYS A 49 218.424 98.945 175.833 1.00 56.33 C ATOM 32 OLYS A 49 217.205 98.904 175.688 1.00 61.95 O ATOM 33 CB LYS A 49 219.74198.923 177.947 1.00 46.86 C ATOM 34 CG LYS A 49 220.349 99.673 179.1031.00 52.81 C ATOM 35 CD LYS A 49 221.201 100.796 178.607 1.00 52.94 CATOM 36 CE LYS A 49 222.255 101.150 179.627 1.00 106.42 C ATOM 37 NZ LYSA 49 223.200 102.166 179.091 1.00 117.49 N ATOM 38 N SER A 50 219.25898.248 175.073 1.00 67.67 N ATOM 39 CA SER A 50 218.768 97.403 173.9941.00 61.19 C ATOM 40 C SER A 50 218.240 96.087 174.484 1.00 62.93 C ATOM41 O SER A 50 218.661 95.592 175.528 1.00 78.88 O ATOM 42 CB SER A 50219.875 97.119 172.994 1.00 59.19 C ATOM 43 OG SER A 50 220.167 98.279172.248 1.00 125.35 O ATOM 44 N GLY A 51 217.318 95.516 173.718 1.0062.13 N ATOM 45 CA GLY A 51 216.773 94.223 174.070 1.00 45.10 C ATOM 46C GLY A 51 217.728 93.155 173.571 1.00 32.66 C ATOM 47 O GLY A 51218.558 93.420 172.709 1.00 72.20 O ATOM 48 N LEU A 52 217.634 91.951174.114 1.00 65.04 N ATOM 49 CA LEU A 52 218.509 90.868 173.671 1.0048.78 C ATOM 50 C LEU A 52 218.154 90.388 172.252 1.00 54.66 C ATOM 51 OLEU A 52 216.986 90.340 171.866 1.00 70.19 O ATOM 52 CB LEU A 52 218.42089.677 174.635 1.00 67.84 C ATOM 53 CG LEU A 52 219.214 88.431 174.2271.00 72.80 C ATOM 54 CD1 LEU A 52 220.719 88.700 174.309 1.00 57.35 CATOM 55 CD2 LEU A 52 218.844 87.283 175.128 1.00 66.63 C ATOM 56 N GLN A53 219.170 90.031 171.477 1.00 68.12 N ATOM 57 CA GLN A 53 218.95889.546 170.125 1.00 54.88 C ATOM 58 C GLN A 53 219.752 88.291 169.8581.00 40.76 C ATOM 59 O GLN A 53 220.978 88.278 169.936 1.00 66.36 O ATOM60 CB GLN A 53 219.372 90.588 169.115 1.00 47.08 C ATOM 61 CG GLN A 53219.428 90.042 167.731 1.00 60.85 C ATOM 62 CD GLN A 53 220.160 90.974166.832 1.00 80.61 C ATOM 63 OE1 GLN A 53 220.236 90.753 165.627 1.0099.15 O ATOM 64 NE2 GLN A 53 220.723 92.031 167.411 1.00 83.94 N ATOM 65N ILE A 54 219.050 87.233 169.508 1.00 60.15 N ATOM 66 CA ILE A 54219.705 85.971 169.238 1.00 52.14 C ATOM 67 C ILE A 54 220.190 85.896167.802 1.00 51.88 C ATOM 68 O ILE A 54 219.433 85.575 166.893 1.0058.79 O ATOM 69 CB ILE A 54 218.743 84.814 169.552 1.00 64.06 C ATOM 70CG1 ILE A 54 218.312 84.924 171.010 1.00 33.87 C ATOM 71 CG2 ILE A 54219.412 83.479 169.315 1.00 39.67 C ATOM 72 CD1 ILE A 54 217.727 83.680171.535 1.00 82.78 C ATOM 73 N LYS A 55 221.464 86.197 167.611 1.0048.72 N ATOM 74 CA LYS A 55 222.064 86.182 166.287 1.00 49.52 C ATOM 75C LYS A 55 222.019 84.808 165.633 1.00 56.72 C ATOM 76 O LYS A 55222.270 83.808 166.283 1.00 70.19 O ATOM 77 CB LYS A 55 223.512 86.661166.383 1.00 51.25 C ATOM 78 CG LYS A 55 223.628 88.111 166.815 1.0049.44 C ATOM 79 CD LYS A 55 225.059 88.584 166.817 1.00 89.37 C ATOM 80CE LYS A 55 225.114 90.085 167.040 1.00 73.14 C ATOM 81 NZ LYS A 55226.521 90.612 167.008 1.00 123.08 N ATOM 82 N LYS A 56 221.710 84.758164.343 1.00 41.46 N ATOM 83 CA LYS A 56 221.644 83.482 163.643 1.0066.57 C ATOM 84 C LYS A 56 222.839 83.118 162.767 1.00 55.67 C ATOM 85 OLYS A 56 223.028 81.945 162.455 1.00 66.55 O ATOM 86 CB LYS A 56 220.37983.413 162.793 1.00 48.95 C ATOM 87 CG LYS A 56 219.242 82.751 163.4861.00 57.91 C ATOM 88 CD LYS A 56 219.248 83.157 164.927 1.00 78.89 CATOM 89 CE LYS A 56 217.907 82.899 165.554 1.00 71.88 C ATOM 90 NZ LYS A56 216.873 83.764 164.935 1.00 82.07 N ATOM 91 N ASN A 57 223.640 84.096162.361 1.00 48.62 N ATOM 92 CA ASN A 57 224.788 83.802 161.508 1.0052.30 C ATOM 93 C ASN A 57 225.851 83.039 162.261 1.00 40.32 C ATOM 94 OASN A 57 225.977 83.197 163.467 1.00 58.36 O ATOM 95 CB ASN A 57 225.38085.083 161.004 1.00 42.83 C ATOM 96 CG ASN A 57 225.743 86.007 162.1231.00 73.68 C ATOM 97 OD1 ASN A 57 224.859 86.577 162.794 1.00 48.52 OATOM 98 ND2 ASN A 57 227.056 86.182 162.340 1.00 36.35 N ATOM 99 N ALA A58 226.627 82.223 161.558 1.00 41.49 N ATOM 100 CA ALA A 58 227.66181.432 162.214 1.00 44.46 C ATOM 101 C ALA A 58 228.613 82.283 163.0401.00 44.31 C ATOM 102 O ALA A 58 229.267 83.173 162.516 1.00 45.45 OATOM 103 CB ALA A 58 228.438 80.641 161.194 1.00 32.48 C ATOM 104 N ILEA 59 228.694 81.998 164.334 1.00 46.98 N ATOM 105 CA ILE A 59 229.56982.753 165.228 1.00 54.79 C ATOM 106 C ILE A 59 231.007 82.870 164.6701.00 63.43 C ATOM 107 O ILE A 59 231.766 83.777 165.035 1.00 47.27 OATOM 108 CB ILE A 59 229.605 82.094 166.638 1.00 53.52 C ATOM 109 CG1ILE A 59 230.284 83.012 167.644 1.00 55.42 C ATOM 110 CG2 ILE A 59230.376 80.791 166.588 1.00 40.15 C ATOM 111 CD1 ILE A 59 230.311 82.446169.038 1.00 57.36 C ATOM 112 N ILE A 60 231.375 81.956 163.777 1.0043.08 N ATOM 113 CA ILE A 60 232.743 81.902 163.274 1.00 57.78 C ATOM114 C ILE A 60 233.086 83.151 162.458 1.00 60.65 C ATOM 115 O ILE A 60234.226 83.590 162.377 1.00 72.20 O ATOM 116 CB ILE A 60 232.866 80.659162.393 1.00 38.44 C ATOM 117 CG1 ILE A 60 231.615 80.513 161.518 1.0050.72 C ATOM 118 CG2 ILE A 60 232.965 79.403 163.277 1.00 49.86 C ATOM119 CD1 ILE A 60 231.798 79.479 160.407 1.00 46.70 C ATOM 120 N ASP A 61232.044 83.704 161.811 1.00 32.79 N ATOM 121 CA ASP A 61 232.254 84.863160.953 1.00 58.81 C ATOM 122 C ASP A 61 232.607 86.117 161.758 1.0050.10 C ATOM 123 O ASP A 61 233.397 86.956 161.346 1.00 78.45 O ATOM 124CB ASP A 61 230.972 85.093 160.149 1.00 36.95 C ATOM 125 CG ASP A 61230.476 83.758 159.608 1.00 91.42 C ATOM 126 OD1 ASP A 61 229.268 83.636159.400 1.00 76.09 O ATOM 127 OD2 ASP A 61 231.293 82.864 159.405 1.00122.26 O ATOM 128 N ASP A 62 231.954 86.250 162.929 1.00 62.40 N ATOM129 CA ASP A 62 232.213 87.422 163.761 1.00 63.46 C ATOM 130 C ASP A 62233.328 87.164 164.778 1.00 43.90 C ATOM 131 O ASP A 62 233.994 88.071165.261 1.00 74.39 O ATOM 132 CB ASP A 62 230.918 87.786 164.492 1.0033.21 C ATOM 133 CG ASP A 62 229.843 88.135 163.473 1.00 76.80 C ATOM134 OD1 ASP A 62 230.116 88.979 162.618 1.00 99.65 O ATOM 135 OD2 ASP A62 228.754 87.573 163.546 1.00 84.30 O ATOM 136 N TYR A 63 233.49385.877 165.132 1.00 44.70 N ATOM 137 CA TYR A 63 234.482 85.539 166.1481.00 62.53 C ATOM 138 C TYR A 63 235.502 84.519 165.641 1.00 58.43 CATOM 139 O TYR A 63 235.388 83.956 164.561 1.00 79.42 O ATOM 140 CB TYRA 63 233.742 84.965 167.358 1.00 50.79 C ATOM 141 CG TYR A 63 233.10086.068 168.120 1.00 44.26 C ATOM 142 CD1 TYR A 63 233.802 86.709 169.1361.00 34.66 C ATOM 143 CD2 TYR A 63 231.800 86.477 167.811 1.00 37.87 CATOM 144 CE1 TYR A 63 233.220 87.758 169.831 1.00 27.94 C ATOM 145 CE2TYR A 63 231.215 87.521 168.512 1.00 34.56 C ATOM 146 CZ TYR A 63231.916 88.158 169.519 1.00 56.82 C ATOM 147 OH TYR A 63 231.353 89.224170.192 1.00 41.90 O ATOM 148 N LYS A 64 236.554 84.325 166.455 1.0069.78 N ATOM 149 CA LYS A 64 237.542 83.306 166.129 1.00 60.63 C ATOM150 C LYS A 64 237.831 82.420 167.340 1.00 67.37 C ATOM 151 O LYS A 64238.259 82.882 168.389 1.00 66.71 O ATOM 152 CB LYS A 64 238.825 84.004165.673 1.00 85.98 C ATOM 153 CG LYS A 64 240.084 83.232 166.078 1.0068.10 C ATOM 154 CD LYS A 64 240.235 81.914 165.315 1.00 96.02 C ATOM155 CE LYS A 64 241.289 80.993 165.941 1.00 159.06 C ATOM 156 NZ LYS A64 241.422 79.778 165.141 1.00 151.29 N ATOM 157 N VAL A 65 237.27181.240 167.130 1.00 54.82 N ATOM 158 CA VAL A 65 237.148 80.288 168.2201.00 65.09 C ATOM 159 C VAL A 65 238.437 79.492 168.298 1.00 65.21 CATOM 160 O VAL A 65 238.945 79.029 167.282 1.00 79.23 O ATOM 161 CB VALA 65 235.960 79.329 168.000 1.00 53.30 C ATOM 162 CG1 VAL A 65 235.94878.263 169.075 1.00 68.94 C ATOM 163 CG2 VAL A 65 234.659 80.104 168.0391.00 50.00 C ATOM 164 N THR A 66 238.971 79.338 169.502 1.00 60.78 NATOM 165 CA THR A 66 240.214 78.602 169.680 1.00 73.39 C ATOM 166 C THRA 66 240.032 77.346 170.515 1.00 76.31 C ATOM 167 O THR A 66 238.96977.107 171.080 1.00 77.40 O ATOM 168 CB THR A 66 241.267 79.462 170.3761.00 65.28 C ATOM 169 OG1 THR A 66 241.150 79.289 171.798 1.00 71.85 OATOM 170 CG2 THR A 66 241.067 80.944 170.016 1.00 64.31 C ATOM 171 N SERA 67 241.092 76.554 170.602 1.00 86.86 N ATOM 172 CA SER A 67 241.06575.318 171.369 1.00 86.84 C ATOM 173 C SER A 67 241.359 75.528 172.8381.00 78.98 C ATOM 174 O SER A 67 241.140 74.631 173.646 1.00 92.44 OATOM 175 CB SER A 67 242.082 74.338 170.818 1.00 77.74 C ATOM 176 OG SERA 67 241.681 73.901 169.540 1.00 131.17 O ATOM 177 N GLN A 68 241.86876.702 173.185 1.00 65.34 N ATOM 178 CA GLN A 68 242.187 76.981 174.5711.00 79.87 C ATOM 179 C GLN A 68 240.955 76.819 175.448 1.00 75.61 CATOM 180 O GLN A 68 239.849 77.197 175.067 1.00 70.47 O ATOM 181 CB GLNA 68 242.760 78.387 174.704 1.00 76.67 C ATOM 182 CG GLN A 68 243.23878.724 176.100 1.00 86.58 C ATOM 183 CD GLN A 68 244.346 79.757 176.0871.00 151.90 C ATOM 184 OE1 GLN A 68 244.745 80.271 177.134 1.00 147.03 OATOM 185 NE2 GLN A 68 244.860 80.060 174.895 1.00 166.06 N ATOM 186 NVAL A 69 241.155 76.244 176.625 1.00 70.23 N ATOM 187 CA VAL A 69240.064 76.013 177.552 1.00 73.71 C ATOM 188 C VAL A 69 240.157 76.908178.775 1.00 65.57 C ATOM 189 O VAL A 69 241.009 76.704 179.639 1.0083.31 O ATOM 190 CB VAL A 69 240.064 74.564 178.023 1.00 69.41 C ATOM191 CG1 VAL A 69 238.896 74.324 178.952 1.00 51.08 C ATOM 192 CG2 VAL A69 240.017 73.648 176.830 1.00 57.48 C ATOM 193 N LEU A 70 239.27677.899 178.847 1.00 74.02 N ATOM 194 CA LEU A 70 239.259 78.816 179.9771.00 88.16 C ATOM 195 C LEU A 70 238.865 78.039 181.227 1.00 94.93 CATOM 196 O LEU A 70 239.171 78.458 182.343 1.00 86.96 O ATOM 197 CB LEUA 70 238.268 79.956 179.718 1.00 80.15 C ATOM 198 CG LEU A 70 238.66081.015 178.680 1.00 66.85 C ATOM 199 CD1 LEU A 70 237.420 81.668 178.0971.00 90.30 C ATOM 200 CD2 LEU A 70 239.553 82.049 179.322 1.00 79.20 CATOM 201 N GLY A 71 238.194 76.903 181.035 1.00 87.34 N ATOM 202 CA GLYA 71 237.781 76.089 182.166 1.00 87.75 C ATOM 203 C GLY A 71 236.70775.077 181.825 1.00 83.77 C ATOM 204 O GLY A 71 236.158 75.111 180.7301.00 84.32 O ATOM 205 N LEU A 72 236.405 74.175 182.757 1.00 88.68 NATOM 206 CA LEU A 72 235.381 73.155 182.534 1.00 82.29 C ATOM 207 C LEUA 72 234.225 73.239 183.509 1.00 73.28 C ATOM 208 O LEU A 72 234.31873.871 184.557 1.00 95.04 O ATOM 209 CB LEU A 72 235.976 71.751 182.6211.00 77.46 C ATOM 210 CG LEU A 72 236.877 71.305 181.473 1.00 94.19 CATOM 211 CD1 LEU A 72 237.217 69.818 181.595 1.00 139.26 C ATOM 212 CD2LEU A 72 236.150 71.568 180.172 1.00 109.03 C ATOM 213 N GLY A 73233.133 72.577 183.158 1.00 77.98 N ATOM 214 CA GLY A 73 231.963 72.592184.013 1.00 71.81 C ATOM 215 C GLY A 73 230.762 71.853 183.446 1.0099.78 C ATOM 216 O GLY A 73 230.843 71.171 182.418 1.00 93.36 O ATOM 217N ILE A 74 229.635 72.004 184.136 1.00 112.58 N ATOM 218 CA ILE A 74228.376 71.365 183.765 1.00 114.50 C ATOM 219 C ILE A 74 228.103 71.259182.270 1.00 110.24 C ATOM 220 O ILE A 74 227.890 70.162 181.750 1.0094.78 O ATOM 221 CB ILE A 74 227.189 72.085 184.439 1.00 120.25 C ATOM222 CG1 ILE A 74 227.145 71.707 185.921 1.00 127.29 C ATOM 223 CG2 ILE A74 225.889 71.745 183.724 1.00 110.29 C ATOM 224 CD1 ILE A 74 225.96872.285 186.673 1.00 178.93 C ATOM 225 N ASN A 75 228.106 72.393 181.5801.00 113.84 N ATOM 226 CA ASN A 75 227.844 72.395 180.148 1.00 108.71 CATOM 227 C ASN A 75 229.044 72.005 179.296 1.00 100.52 C ATOM 228 O ASNA 75 228.935 71.858 178.079 1.00 102.09 O ATOM 229 CB ASN A 75 227.30373.756 179.736 1.00 95.22 C ATOM 230 CG ASN A 75 225.898 73.967 180.2251.00 84.98 C ATOM 231 OD1 ASN A 75 224.966 73.315 179.749 1.00 88.40 OATOM 232 ND2 ASN A 75 225.730 74.858 181.199 1.00 112.62 N ATOM 233 NGLY A 76 230.186 71.820 179.940 1.00 99.31 N ATOM 234 CA GLY A 76231.360 71.428 179.198 1.00 93.20 C ATOM 235 C GLY A 76 232.489 72.428179.282 1.00 96.95 C ATOM 236 O GLY A 76 232.666 73.117 180.285 1.00101.12 O ATOM 237 N LYS A 77 233.258 72.506 178.205 1.00 82.50 N ATOM238 CA LYS A 77 234.386 73.409 178.146 1.00 79.18 C ATOM 239 C LYS A 77233.986 74.820 177.757 1.00 66.93 C ATOM 240 O LYS A 77 233.189 75.025176.852 1.00 79.35 O ATOM 241 CB LYS A 77 235.410 72.872 177.160 1.0078.70 C ATOM 242 N VAL A 78 234.523 75.794 178.470 1.00 57.33 N ATOM 243CA VAL A 78 234.267 77.179 178.150 1.00 49.75 C ATOM 244 C VAL A 78235.512 77.620 177.410 1.00 79.35 C ATOM 245 O VAL A 78 236.545 77.888178.028 1.00 64.43 O ATOM 246 CB VAL A 78 234.138 78.020 179.386 1.0054.95 C ATOM 247 CG1 VAL A 78 234.267 79.482 179.023 1.00 45.00 C ATOM248 CG2 VAL A 78 232.810 77.742 180.029 1.00 52.90 C ATOM 249 N LEU A 79235.414 77.692 176.087 1.00 70.72 N ATOM 250 CA LEU A 79 236.538 78.073175.245 1.00 61.89 C ATOM 251 C LEU A 79 236.825 79.563 175.185 1.0064.76 C ATOM 252 O LEU A 79 235.930 80.394 175.360 1.00 63.50 O ATOM 253CB LEU A 79 236.292 77.599 173.825 1.00 51.24 C ATOM 254 CG LEU A 79235.907 76.149 173.599 1.00 64.90 C ATOM 255 CD1 LEU A 79 235.543 75.983172.139 1.00 49.75 C ATOM 256 CD2 LEU A 79 237.052 75.236 173.978 1.0080.17 C ATOM 257 N GLN A 80 238.090 79.889 174.919 1.00 72.17 N ATOM 258CA GLN A 80 238.505 81.274 174.765 1.00 77.62 C ATOM 259 C GLN A 80238.376 81.604 173.288 1.00 66.11 C ATOM 260 O GLN A 80 238.775 80.813172.437 1.00 64.63 O ATOM 261 CB GLN A 80 239.955 81.486 175.167 1.0052.97 C ATOM 262 CG GLN A 80 240.390 82.913 174.863 1.00 93.41 C ATOM263 CD GLN A 80 241.715 83.310 175.494 1.00 115.14 C ATOM 264 OE1 GLN A80 242.038 84.502 175.567 1.00 116.65 O ATOM 265 NE2 GLN A 80 242.49082.322 175.949 1.00 117.09 N ATOM 266 N ILE A 81 237.792 82.754 172.9821.00 57.33 N ATOM 267 CA ILE A 81 237.629 83.150 171.596 1.00 57.32 CATOM 268 C ILE A 81 238.109 84.571 171.400 1.00 54.90 C ATOM 269 O ILE A81 238.310 85.320 172.362 1.00 61.04 O ATOM 270 CB ILE A 81 236.16083.072 171.114 1.00 62.17 C ATOM 271 CG1 ILE A 81 235.289 84.050 171.8971.00 51.03 C ATOM 272 CG2 ILE A 81 235.628 81.679 171.285 1.00 42.99 CATOM 273 CD1 ILE A 81 233.852 84.088 171.391 1.00 59.46 C ATOM 274 N PHEA 82 238.287 84.930 170.131 1.00 70.25 N ATOM 275 CA PHE A 82 238.75386.251 169.763 1.00 55.36 C ATOM 276 C PHE A 82 237.815 86.911 168.7871.00 62.80 C ATOM 277 O PHE A 82 237.287 86.260 167.874 1.00 65.67 OATOM 278 CB PHE A 82 240.145 86.152 169.152 1.00 42.74 C ATOM 279 CG PHEA 82 241.197 85.862 170.151 1.00 59.25 C ATOM 280 CD1 PHE A 82 241.63986.857 170.999 1.00 44.66 C ATOM 281 CD2 PHE A 82 241.687 84.576 170.3141.00 37.34 C ATOM 282 CE1 PHE A 82 242.552 86.576 171.999 1.00 57.81 CATOM 283 CE2 PHE A 82 242.604 84.286 171.315 1.00 54.82 C ATOM 284 CZPHE A 82 243.032 85.287 172.156 1.00 58.83 C ATOM 285 N ASN A 83 237.59288.205 168.997 1.00 52.27 N ATOM 286 CA ASN A 83 236.746 88.954 168.1041.00 66.04 C ATOM 287 C ASN A 83 237.605 89.492 166.971 1.00 53.09 CATOM 288 O ASN A 83 238.453 90.346 167.171 1.00 64.85 O ATOM 289 CB ASNA 83 236.093 90.099 168.830 1.00 47.48 C ATOM 290 CG ASN A 83 235.21490.902 167.924 1.00 67.70 C ATOM 291 OD1 ASN A 83 235.689 91.744 167.1601.00 72.99 O ATOM 292 ND2 ASN A 83 233.915 90.632 167.977 1.00 70.38 NATOM 293 N LYS A 84 237.383 88.974 165.774 1.00 63.87 N ATOM 294 CA LYSA 84 238.133 89.376 164.590 1.00 68.20 C ATOM 295 C LYS A 84 238.36690.876 164.450 1.00 61.51 C ATOM 296 O LYS A 84 239.492 91.340 164.5371.00 74.81 O ATOM 297 CB LYS A 84 237.412 88.859 163.345 1.00 55.26 CATOM 298 CG LYS A 84 237.312 87.343 163.283 1.00 33.38 C ATOM 299 CD LYSA 84 236.425 86.918 162.154 1.00 30.89 C ATOM 300 CE LYS A 84 236.75985.521 161.719 1.00 46.41 C ATOM 301 NZ LYS A 84 236.028 85.182 160.4761.00 72.55 N ATOM 302 N ARG A 85 237.290 91.622 164.235 1.00 63.12 NATOM 303 CA ARG A 85 237.359 93.066 164.043 1.00 51.69 C ATOM 304 C ARGA 85 237.905 93.905 165.183 1.00 58.02 C ATOM 305 O ARG A 85 238.19795.075 164.991 1.00 86.09 O ATOM 306 CB ARG A 85 235.978 93.589 163.6471.00 60.23 C ATOM 307 CG ARG A 85 235.862 95.086 163.509 1.00 76.93 CATOM 308 CD ARG A 85 235.516 95.725 164.835 1.00 84.92 C ATOM 309 NE ARGA 85 234.121 96.140 164.907 1.00 93.42 N ATOM 310 CZ ARG A 85 233.54196.634 165.997 1.00 103.30 C ATOM 311 NH1 ARG A 85 234.224 96.777167.128 1.00 54.02 N ATOM 312 NH2 ARG A 85 232.268 96.998 165.949 1.00119.53 N ATOM 313 N THR A 86 238.050 93.339 166.370 1.00 64.72 N ATOM314 CA THR A 86 238.565 94.123 167.489 1.00 54.27 C ATOM 315 C THR A 86239.717 93.398 168.140 1.00 52.40 C ATOM 316 O THR A 86 240.520 93.980168.856 1.00 57.78 O ATOM 317 CB THR A 86 237.468 94.392 168.536 1.0058.63 C ATOM 318 OG1 THR A 86 236.471 95.246 167.968 1.00 54.24 O ATOM319 CG2 THR A 86 238.045 95.084 169.743 1.00 98.48 C ATOM 320 N GLN A 87239.772 92.106 167.883 1.00 54.70 N ATOM 321 CA GLN A 87 240.819 91.260168.397 1.00 49.69 C ATOM 322 C GLN A 87 240.875 91.131 169.927 1.0063.54 C ATOM 323 O GLN A 87 241.936 90.881 170.491 1.00 78.48 O ATOM 324CB GLN A 87 242.148 91.761 167.854 1.00 44.04 C ATOM 325 CG GLN A 87243.145 90.675 167.561 1.00 87.54 C ATOM 326 CD GLN A 87 242.722 89.811166.413 1.00 70.54 C ATOM 327 OE1 GLN A 87 243.394 88.840 166.083 1.0095.76 O ATOM 328 NE2 GLN A 87 241.601 90.157 165.789 1.00 75.13 N ATOM329 N GLU A 88 239.746 91.291 170.608 1.00 57.33 N ATOM 330 CA GLU A 88239.751 91.135 172.060 1.00 69.04 C ATOM 331 C GLU A 88 239.352 89.723172.442 1.00 68.23 C ATOM 332 O GLU A 88 238.622 89.060 171.702 1.0070.57 O ATOM 333 CB GLU A 88 238.816 92.138 172.708 1.00 73.60 C ATOM334 CG GLU A 88 239.297 93.538 172.480 1.00 111.93 C ATOM 335 CD GLU A88 240.559 93.845 173.228 1.00 130.01 C ATOM 336 OE1 GLU A 88 240.72893.298 174.344 1.00 151.01 O ATOM 337 OE2 GLU A 88 241.376 94.634172.698 1.00 169.07 O ATOM 338 N LYS A 89 239.837 89.261 173.592 1.0080.21 N ATOM 339 CA LYS A 89 239.543 87.904 174.047 1.00 67.45 C ATOM340 C LYS A 89 238.171 87.815 174.709 1.00 67.74 C ATOM 341 O LYS A 89237.719 88.752 175.355 1.00 71.95 O ATOM 342 CB LYS A 89 240.632 87.424175.013 1.00 46.26 C ATOM 343 N PHE A 90 237.504 86.681 174.537 1.0068.69 N ATOM 344 CA PHE A 90 236.195 86.481 175.128 1.00 63.31 C ATOM345 C PHE A 90 235.957 85.019 175.477 1.00 70.09 C ATOM 346 O PHE A 90236.571 84.121 174.892 1.00 76.39 O ATOM 347 CB PHE A 90 235.100 86.935174.169 1.00 52.49 C ATOM 348 CG PHE A 90 234.975 88.434 174.031 1.0061.34 C ATOM 349 CD1 PHE A 90 235.523 89.091 172.952 1.00 63.36 C ATOM350 CD2 PHE A 90 234.238 89.170 174.942 1.00 60.92 C ATOM 351 CE1 PHE A90 235.330 90.429 172.781 1.00 38.41 C ATOM 352 CE2 PHE A 90 234.04890.529 174.762 1.00 57.58 C ATOM 353 CZ PHE A 90 234.591 91.155 173.6831.00 50.60 C ATOM 354 N ALA A 91 235.055 84.788 176.431 1.00 67.69 NATOM 355 CA ALA A 91 234.692 83.444 176.853 1.00 44.28 C ATOM 356 C ALAA 91 233.480 83.000 176.052 1.00 45.25 C ATOM 357 O ALA A 91 232.56983.788 175.795 1.00 60.93 O ATOM 358 CB ALA A 91 234.360 83.443 178.3021.00 52.11 C ATOM 359 N LEU A 92 233.477 81.736 175.653 1.00 48.94 NATOM 360 CA LEU A 92 232.381 81.179 174.882 1.00 50.98 C ATOM 361 C LEUA 92 231.771 79.995 175.610 1.00 66.99 C ATOM 362 O LEU A 92 232.46779.045 175.939 1.00 82.82 O ATOM 363 CB LEU A 92 232.886 80.710 173.5361.00 39.13 C ATOM 364 CG LEU A 92 231.838 79.857 172.839 1.00 52.58 CATOM 365 CD1 LEU A 92 230.636 80.726 172.548 1.00 48.33 C ATOM 366 CD2LEU A 92 232.404 79.256 171.557 1.00 58.60 C ATOM 367 N LYS A 93 230.46980.053 175.856 1.00 66.30 N ATOM 368 CA LYS A 93 229.650 79.022 176.4861.00 62.11 C ATOM 369 C LYS A 93 228.624 78.440 175.508 1.00 61.26 CATOM 370 O LYS A 93 227.945 79.148 174.774 1.00 77.67 O ATOM 371 CB LYSA 93 228.936 79.645 177.689 1.00 79.47 C ATOM 372 CG LYS A 93 228.64078.617 178.785 1.00 69.18 C ATOM 373 CD LYS A 93 227.896 79.231 179.9741.00 74.19 C ATOM 374 CE LYS A 93 227.871 78.303 181.194 1.00 107.78 CATOM 375 NZ LYS A 93 228.986 78.626 182.083 1.00 92.73 N ATOM 376 N META 94 228.551 77.095 175.489 1.00 53.94 N ATOM 377 CA MET A 94 227.63976.436 174.560 1.00 63.34 C ATOM 378 C MET A 94 226.517 75.693 175.2901.00 67.05 C ATOM 379 O MET A 94 226.740 74.805 176.101 1.00 77.93 OATOM 380 CB MET A 94 228.447 75.451 173.713 1.00 54.57 C ATOM 381 CG META 94 229.751 76.056 173.188 1.00 66.95 C ATOM 382 SD MET A 94 230.85574.810 172.511 1.00 128.32 S ATOM 383 CE MET A 94 231.935 75.912 171.5871.00 110.84 C ATOM 384 N LEU A 95 225.273 76.116 174.999 1.00 76.30 NATOM 385 CA LEU A 95 224.126 75.468 175.626 1.00 47.37 C ATOM 386 C LEUA 95 223.284 74.703 174.601 1.00 61.07 C ATOM 387 O LEU A 95 223.14375.102 173.452 1.00 66.44 O ATOM 388 CB LEU A 95 223.276 76.549 176.2951.00 56.39 C ATOM 389 CG LEU A 95 224.057 77.342 177.346 1.00 78.66 CATOM 390 CD1 LEU A 95 223.138 78.105 178.302 1.00 71.39 C ATOM 391 CD2LEU A 95 224.941 76.453 178.221 1.00 68.93 C ATOM 392 N GLN A 96 222.57473.621 174.890 1.00 60.77 N ATOM 393 CA GLN A 96 221.662 73.075 173.9061.00 63.29 C ATOM 394 C GLN A 96 220.493 74.049 173.876 1.00 63.17 CATOM 395 O GLN A 96 219.998 74.481 174.915 1.00 63.30 O ATOM 396 CB GLNA 96 221.193 71.700 174.326 1.00 70.27 C ATOM 397 N ASP A 97 220.05674.415 172.686 1.00 59.87 N ATOM 398 CA ASP A 97 218.950 75.343 172.5891.00 58.98 C ATOM 399 C ASP A 97 217.669 74.640 173.022 1.00 73.48 CATOM 400 O ASP A 97 217.277 73.626 172.448 1.00 75.22 O ATOM 401 CB ASPA 97 218.831 75.843 171.157 1.00 68.14 C ATOM 402 CG ASP A 97 217.93377.038 171.045 1.00 73.65 C ATOM 403 OD1 ASP A 97 217.516 77.567 172.0961.00 68.14 O ATOM 404 OD2 ASP A 97 217.647 77.451 169.906 1.00 80.65 OATOM 405 N CYS A 98 217.023 75.189 174.041 1.00 67.24 N ATOM 406 CA CYSA 98 215.801 74.626 174.586 1.00 52.14 C ATOM 407 C CYS A 98 215.25875.697 175.510 1.00 75.99 C ATOM 408 O CYS A 98 216.018 76.483 176.0681.00 86.44 O ATOM 409 CB CYS A 98 216.122 73.415 175.421 1.00 52.47 CATOM 410 SG CYS A 98 216.922 73.906 176.969 1.00 68.25 S ATOM 411 N PROA 99 213.939 75.722 175.717 1.00 81.29 N ATOM 412 CA PRO A 99 213.28376.705 176.578 1.00 85.87 C ATOM 413 C PRO A 99 213.996 77.083 177.8681.00 82.76 C ATOM 414 O PRO A 99 214.153 78.268 178.149 1.00 67.85 OATOM 415 CB PRO A 99 211.926 76.073 176.821 1.00 94.07 C ATOM 416 CG PROA 99 211.642 75.452 175.492 1.00 91.19 C ATOM 417 CD PRO A 99 212.95974.756 175.201 1.00 93.97 C ATOM 418 N LYS A 100 214.435 76.098 178.6481.00 67.78 N ATOM 419 CA LYS A 100 215.100 76.408 179.912 1.00 66.60 CATOM 420 C LYS A 100 216.341 77.236 179.702 1.00 80.42 C ATOM 421 O LYSA 100 216.622 78.158 180.472 1.00 69.23 O ATOM 422 CB LYS A 100 215.47375.088 180.595 1.00 84.50 C ATOM 423 N ALA A 101 217.085 76.885 178.6561.00 76.33 N ATOM 424 CA ALA A 101 218.320 77.570 178.319 1.00 66.38 CATOM 425 C ALA A 101 218.055 79.028 177.964 1.00 67.87 C ATOM 426 O ALAA 101 218.639 79.922 178.563 1.00 70.18 O ATOM 427 CB ALA A 101 219.02276.849 177.175 1.00 55.84 C ATOM 428 N ARG A 102 217.163 79.277 177.0141.00 63.42 N ATOM 429 CA ARG A 102 216.852 80.646 176.622 1.00 64.85 CATOM 430 C ARG A 102 216.409 81.491 177.812 1.00 68.04 C ATOM 431 O ARGA 102 216.523 82.715 177.785 1.00 87.14 O ATOM 432 CB ARG A 102 215.76480.665 175.541 1.00 58.29 C ATOM 433 CG ARG A 102 216.118 79.877 174.2901.00 87.87 C ATOM 434 CD ARG A 102 215.938 80.688 173.003 1.00 113.57 CATOM 435 NE ARG A 102 216.532 80.000 171.853 1.00 149.75 N ATOM 436 CZARG A 102 216.645 80.517 170.626 1.00 161.97 C ATOM 437 NH1 ARG A 102216.200 81.742 170.361 1.00 169.18 N ATOM 438 NH2 ARG A 102 217.20979.808 169.658 1.00 123.53 N ATOM 439 N ARG A 103 215.900 80.848 178.8561.00 68.81 N ATOM 440 CA ARG A 103 215.456 81.579 180.040 1.00 80.79 CATOM 441 C ARG A 103 216.664 82.065 180.812 1.00 74.21 C ATOM 442 O ARGA 103 216.757 83.229 181.192 1.00 79.04 O ATOM 443 CB ARG A 103 214.62080.684 180.943 1.00 100.01 C ATOM 444 CG ARG A 103 214.334 81.302182.299 1.00 140.54 C ATOM 445 CD ARG A 103 213.451 80.396 183.152 1.00186.85 C ATOM 446 NE ARG A 103 214.159 79.246 183.721 1.00 183.90 N ATOM447 CZ ARG A 103 213.851 77.974 183.469 1.00 163.96 C ATOM 448 NH1 ARG A103 212.848 77.669 182.641 1.00 83.48 N ATOM 449 NH2 ARG A 103 214.52377.008 184.082 1.00 172.74 N ATOM 450 N GLU A 104 217.584 81.143 181.0471.00 62.81 N ATOM 451 CA GLU A 104 218.821 81.431 181.750 1.00 74.19 CATOM 452 C GLU A 104 219.481 82.643 181.094 1.00 76.22 C ATOM 453 O GLUA 104 219.642 83.699 181.711 1.00 64.24 O ATOM 454 CB GLU A 104 219.72280.210 181.633 1.00 84.57 C ATOM 455 CG GLU A 104 221.043 80.286 182.3421.00 113.52 C ATOM 456 CD GLU A 104 221.832 79.002 182.167 1.00 109.63 CATOM 457 OE1 GLU A 104 222.923 78.879 182.757 1.00 143.02 O ATOM 458 OE2GLU A 104 221.358 78.112 181.430 1.00 133.75 O ATOM 459 N VAL A 105219.845 82.462 179.828 1.00 72.46 N ATOM 460 CA VAL A 105 220.488 83.485179.010 1.00 67.75 C ATOM 461 C VAL A 105 219.830 84.846 179.164 1.0071.54 C ATOM 462 O VAL A 105 220.506 85.851 179.366 1.00 75.44 O ATOM463 CB VAL A 105 220.450 83.096 177.524 1.00 70.64 C ATOM 464 CG1 VAL A105 221.057 84.183 176.695 1.00 60.07 C ATOM 465 CG2 VAL A 105 221.19681.796 177.312 1.00 75.62 C ATOM 466 N GLU A 106 218.511 84.879 179.0551.00 56.08 N ATOM 467 CA GLU A 106 217.795 86.126 179.198 1.00 58.27 CATOM 468 C GLU A 106 218.087 86.715 180.569 1.00 62.94 C ATOM 469 O GLUA 106 218.474 87.873 180.682 1.00 69.82 O ATOM 470 CB GLU A 106 216.30185.893 179.081 1.00 60.31 C ATOM 471 CG GLU A 106 215.588 86.837 178.1561.00 75.77 C ATOM 472 CD GLU A 106 215.465 86.257 176.774 1.00 90.69 CATOM 473 OE1 GLU A 106 216.490 86.211 176.072 1.00 127.18 O ATOM 474 OE2GLU A 106 214.352 85.823 176.393 1.00 144.79 O ATOM 475 N LEU A 107217.896 85.921 181.617 1.00 59.54 N ATOM 476 CA LEU A 107 218.132 86.409182.964 1.00 60.25 C ATOM 477 C LEU A 107 219.503 87.050 183.098 1.0079.14 C ATOM 478 O LEU A 107 219.649 88.148 183.623 1.00 55.44 O ATOM479 CB LEU A 107 218.013 85.269 183.967 1.00 82.80 C ATOM 480 CG LEU A107 216.618 84.741 184.297 1.00 74.03 C ATOM 481 CD1 LEU A 107 216.74083.679 185.368 1.00 68.79 C ATOM 482 CD2 LEU A 107 215.731 85.866184.783 1.00 64.45 C ATOM 483 N HIS A 108 220.514 86.351 182.614 1.0068.47 N ATOM 484 CA HIS A 108 221.875 86.840 182.691 1.00 60.51 C ATOM485 C HIS A 108 222.058 88.075 181.819 1.00 51.27 C ATOM 486 O HIS A 108222.721 89.035 182.208 1.00 70.90 O ATOM 487 CB HIS A 108 222.817 85.732182.252 1.00 74.21 C ATOM 488 CG HIS A 108 224.261 86.055 182.447 1.0058.20 C ATOM 489 ND1 HIS A 108 225.260 85.149 182.176 1.00 56.15 N ATOM490 CD2 HIS A 108 224.875 87.176 182.891 1.00 56.76 C ATOM 491 CE1 HIS A108 226.429 85.699 182.446 1.00 52.10 C ATOM 492 NE2 HIS A 108 226.22486.928 182.881 1.00 52.02 N ATOM 493 N TRP A 109 221.480 88.046 180.6281.00 57.29 N ATOM 494 CA TRP A 109 221.633 89.219 179.779 1.00 60.60 CATOM 495 C TRP A 109 220.952 90.440 180.401 1.00 49.86 C ATOM 496 O TRPA 109 221.365 91.583 180.231 1.00 77.39 O ATOM 497 CB TRP A 109 221.00088.905 178.423 1.00 58.21 C ATOM 498 CG TRP A 109 220.711 90.158 177.6911.00 62.11 C ATOM 499 CD1 TRP A 109 219.493 90.874 177.686 1.00 77.34 CATOM 500 CD2 TRP A 109 221.631 90.902 176.855 1.00 77.76 C ATOM 501 NE1TRP A 109 219.557 92.006 176.931 1.00 78.01 N ATOM 502 CE2 TRP A 109220.933 92.041 176.375 1.00 87.90 C ATOM 503 CE3 TRP A 109 222.95490.700 176.473 1.00 105.10 C ATOM 504 CZ2 TRP A 109 221.572 92.943175.540 1.00 127.04 C ATOM 505 CZ3 TRP A 109 223.594 91.606 175.640 1.00107.99 C ATOM 506 CH2 TRP A 109 222.895 92.731 175.167 1.00 111.87 CATOM 507 N ARG A 110 219.845 90.163 181.113 1.00 63.80 N ATOM 508 CA ARGA 110 219.102 91.243 181.753 1.00 60.39 C ATOM 509 C ARG A 110 219.80791.738 183.019 1.00 56.74 C ATOM 510 O ARG A 110 219.416 92.713 183.6481.00 72.39 O ATOM 511 CB ARG A 110 217.714 90.704 182.108 1.00 55.11 CATOM 512 CG ARG A 110 216.630 91.781 182.038 1.00 70.18 C ATOM 513 CDARG A 110 215.241 91.177 181.803 1.00 129.88 C ATOM 514 NE ARG A 110214.340 91.500 182.914 1.00 98.93 N ATOM 515 CZ ARG A 110 214.072 90.515183.794 1.00 97.18 C ATOM 516 NH1 ARG A 110 213.537 90.802 184.969 1.00106.39 N ATOM 517 NH2 ARG A 110 214.325 89.245 183.464 1.00 84.21 N ATOM518 N ALA A 111 220.864 90.997 183.409 1.00 61.92 N ATOM 519 CA ALA A111 221.578 91.358 184.628 1.00 62.44 C ATOM 520 C ALA A 111 223.02391.776 184.343 1.00 78.96 C ATOM 521 O ALA A 111 223.808 92.067 185.2371.00 57.41 O ATOM 522 CB ALA A 111 221.559 90.151 185.567 1.00 53.16 CATOM 523 N SER A 112 223.377 91.758 183.046 1.00 76.17 N ATOM 524 CA SERA 112 224.737 92.128 182.670 1.00 71.16 C ATOM 525 C SER A 112 224.97993.632 182.825 1.00 64.82 C ATOM 526 O SER A 112 226.104 94.113 182.8061.00 63.34 O ATOM 527 CB SER A 112 224.963 91.711 181.216 1.00 45.39 CATOM 528 OG SER A 112 224.992 90.282 181.135 1.00 81.43 O ATOM 529 N GLNA 113 223.918 94.403 182.989 1.00 73.53 N ATOM 530 CA GLN A 113 224.07195.835 183.163 1.00 77.61 C ATOM 531 C GLN A 113 224.644 96.132 184.5451.00 70.17 C ATOM 532 O GLN A 113 224.884 97.280 184.884 1.00 88.91 OATOM 533 CB GLN A 113 222.716 96.537 183.006 1.00 79.72 C ATOM 534 CGGLN A 113 221.947 96.134 181.754 1.00 117.37 C ATOM 535 CD GLN A 113222.616 96.582 180.448 1.00 132.38 C ATOM 536 OE1 GLN A 113 222.30596.060 179.363 1.00 85.14 O ATOM 537 NE2 GLN A 113 223.522 97.558180.542 1.00 73.13 N ATOM 538 N CYS A 114 224.854 95.098 185.348 1.0077.28 N ATOM 539 CA CYS A 114 225.395 95.280 186.692 1.00 65.33 C ATOM540 C CYS A 114 226.896 95.029 186.750 1.00 66.81 C ATOM 541 O CYS A 114227.365 93.910 186.553 1.00 70.94 O ATOM 542 CB CYS A 114 224.699 94.346187.679 1.00 68.99 C ATOM 543 SG CYS A 114 225.606 94.158 189.225 1.0070.19 S ATOM 544 N PRO A 115 227.665 96.073 187.058 1.00 57.36 N ATOM545 CA PRO A 115 229.123 96.082 187.171 1.00 46.75 C ATOM 546 C PRO A115 229.770 94.790 187.650 1.00 60.90 C ATOM 547 O PRO A 115 230.73594.315 187.046 1.00 79.32 O ATOM 548 CB PRO A 115 229.382 97.226 188.1401.00 46.54 C ATOM 549 CG PRO A 115 228.325 98.199 187.764 1.00 65.08 CATOM 550 CD PRO A 115 227.093 97.329 187.569 1.00 53.52 C ATOM 551 N HISA 116 229.242 94.217 188.726 1.00 50.47 N ATOM 552 CA HIS A 116 229.82793.015 189.294 1.00 62.43 C ATOM 553 C HIS A 116 229.336 91.675 188.7871.00 62.85 C ATOM 554 O HIS A 116 229.654 90.634 189.366 1.00 86.03 OATOM 555 CB HIS A 116 229.709 93.085 190.804 1.00 66.18 C ATOM 556 CGHIS A 116 230.547 94.163 191.405 1.00 74.19 C ATOM 557 ND1 HIS A 116231.923 94.104 191.422 1.00 58.68 N ATOM 558 CD2 HIS A 116 230.21495.360 191.945 1.00 50.93 C ATOM 559 CE1 HIS A 116 232.404 95.220191.945 1.00 106.08 C ATOM 560 NE2 HIS A 116 231.386 95.999 192.269 1.0097.40 N ATOM 561 N ILE A 117 228.574 91.693 187.703 1.00 58.07 N ATOM562 CA ILE A 117 228.075 90.472 187.097 1.00 51.42 C ATOM 563 C ILE A117 228.790 90.375 185.769 1.00 57.68 C ATOM 564 O ILE A 117 228.91291.381 185.056 1.00 72.95 O ATOM 565 CB ILE A 117 226.586 90.572 186.8481.00 60.05 C ATOM 566 CG1 ILE A 117 225.867 90.676 188.193 1.00 65.42 CATOM 567 CG2 ILE A 117 226.111 89.371 186.050 1.00 56.83 C ATOM 568 CD1ILE A 117 224.420 91.064 188.100 1.00 76.80 C ATOM 569 N VAL A 118229.275 89.188 185.427 1.00 60.67 N ATOM 570 CA VAL A 118 229.979 89.028184.160 1.00 56.50 C ATOM 571 C VAL A 118 229.008 89.416 183.044 1.0062.86 C ATOM 572 O VAL A 118 227.844 89.008 183.071 1.00 61.80 O ATOM573 CB VAL A 118 230.469 87.583 183.975 1.00 59.65 C ATOM 574 CG1 VAL A118 229.288 86.642 183.812 1.00 58.62 C ATOM 575 CG2 VAL A 118 231.38987.503 182.776 1.00 72.68 C ATOM 576 N ARG A 119 229.483 90.204 182.0751.00 59.87 N ATOM 577 CA ARG A 119 228.635 90.694 180.988 1.00 63.38 CATOM 578 C ARG A 119 228.551 89.821 179.763 1.00 57.05 C ATOM 579 O ARGA 119 229.546 89.276 179.314 1.00 63.34 O ATOM 580 CB ARG A 119 229.08392.079 180.566 1.00 37.88 C ATOM 581 N ILE A 120 227.348 89.698 179.2171.00 52.84 N ATOM 582 CA ILE A 120 227.135 88.928 178.002 1.00 55.21 CATOM 583 C ILE A 120 227.227 89.914 176.847 1.00 56.02 C ATOM 584 O ILEA 120 226.375 90.801 176.719 1.00 61.19 O ATOM 585 CB ILE A 120 225.74288.298 177.985 1.00 63.39 C ATOM 586 CG1 ILE A 120 225.707 87.100178.931 1.00 59.03 C ATOM 587 CG2 ILE A 120 225.368 87.898 176.570 1.0045.00 C ATOM 588 CD1 ILE A 120 224.390 86.384 178.896 1.00 71.76 C ATOM589 N VAL A 121 228.259 89.766 176.013 1.00 69.54 N ATOM 590 CA VAL A121 228.440 90.647 174.863 1.00 53.54 C ATOM 591 C VAL A 121 227.43290.354 173.743 1.00 53.36 C ATOM 592 O VAL A 121 226.823 91.254 173.1731.00 67.91 O ATOM 593 CB VAL A 121 229.875 90.482 174.354 1.00 52.53 CATOM 594 CG1 VAL A 121 230.153 91.471 173.224 1.00 91.60 C ATOM 595 CG2VAL A 121 230.854 90.737 175.486 1.00 71.15 C ATOM 596 N ASP A 122227.299 89.060 173.389 1.00 45.30 N ATOM 597 CA ASP A 122 226.149 88.676172.567 1.00 76.20 C ATOM 598 C ASP A 122 225.984 87.152 172.459 1.0052.20 C ATOM 599 O ASP A 122 226.852 86.378 172.842 1.00 62.83 O ATOM600 CB ASP A 122 226.337 89.280 171.173 1.00 81.39 C ATOM 601 CG ASP A122 227.687 88.850 170.615 1.00 72.92 C ATOM 602 OD1 ASP A 122 228.22487.860 171.115 1.00 121.42 O ATOM 603 OD2 ASP A 122 228.183 89.500169.698 1.00 55.20 O ATOM 604 N VAL A 123 224.777 86.769 171.999 1.0050.00 N ATOM 605 CA VAL A 123 224.398 85.361 171.982 1.00 55.40 C ATOM606 C VAL A 123 223.988 84.917 170.574 1.00 50.66 C ATOM 607 O VAL A 123223.350 85.650 169.830 1.00 50.76 O ATOM 608 CB VAL A 123 223.215 85.196172.943 1.00 45.71 C ATOM 609 CG1 VAL A 123 222.819 83.726 173.058 1.0066.35 C ATOM 610 CG2 VAL A 123 223.581 85.730 174.316 1.00 57.34 C ATOM611 N TYR A 124 224.384 83.682 170.271 1.00 54.76 N ATOM 612 CA TYR A124 224.108 83.092 168.964 1.00 59.15 C ATOM 613 C TYR A 124 223.28681.813 169.047 1.00 61.11 C ATOM 614 O TYR A 124 223.356 81.063 170.0211.00 77.18 O ATOM 615 CB TYR A 124 225.412 82.768 168.216 1.00 54.42 CATOM 616 CG TYR A 124 226.292 83.973 167.929 1.00 64.97 C ATOM 617 CD1TYR A 124 227.031 84.574 168.941 1.00 36.17 C ATOM 618 CD2 TYR A 124226.352 84.532 166.649 1.00 68.56 C ATOM 619 CE1 TYR A 124 227.79385.681 168.700 1.00 56.00 C ATOM 620 CE2 TYR A 124 227.117 85.653166.393 1.00 49.47 C ATOM 621 CZ TYR A 124 227.835 86.220 167.428 1.0051.06 C ATOM 622 OH TYR A 124 228.608 87.330 167.202 1.00 71.62 O ATOM623 N GLU A 125 222.497 81.576 168.010 1.00 65.81 N ATOM 624 CA GLU A125 221.697 80.374 167.910 1.00 49.40 C ATOM 625 C GLU A 125 222.16479.747 166.601 1.00 50.33 C ATOM 626 O GLU A 125 221.610 80.007 165.5351.00 63.99 O ATOM 627 CB GLU A 125 220.210 80.728 167.851 1.00 56.44 CATOM 628 CG GLU A 125 219.285 79.525 167.952 1.00 64.02 C ATOM 629 CDGLU A 125 218.600 79.176 166.637 1.00 103.79 C ATOM 630 OE1 GLU A 125217.665 79.900 166.223 1.00 113.21 O ATOM 631 OE2 GLU A 125 219.00278.172 166.009 1.00 118.03 O ATOM 632 N ASN A 126 223.217 78.948 166.6751.00 57.09 N ATOM 633 CA ASN A 126 223.752 78.304 165.480 1.00 63.39 CATOM 634 C ASN A 126 223.370 76.842 165.453 1.00 68.35 C ATOM 635 O ASNA 126 222.770 76.334 166.393 1.00 78.79 O ATOM 636 CB ASN A 126 225.27178.413 165.451 1.00 53.33 C ATOM 637 CG ASN A 126 225.738 79.824 165.2371.00 50.86 C ATOM 638 OD1 ASN A 126 226.934 80.105 165.254 1.00 53.47 OATOM 639 ND2 ASN A 126 224.786 80.732 165.026 1.00 57.45 N ATOM 640 NLEU A 127 223.729 76.167 164.370 1.00 72.36 N ATOM 641 CA LEU A 127223.431 74.753 164.222 1.00 56.01 C ATOM 642 C LEU A 127 224.712 73.983164.467 1.00 63.01 C ATOM 643 O LEU A 127 225.637 74.025 163.663 1.0079.72 O ATOM 644 CB LEU A 127 222.926 74.467 162.817 1.00 57.56 C ATOM645 CG LEU A 127 221.810 73.443 162.741 1.00 56.21 C ATOM 646 CD1 LEU A127 220.660 73.944 163.564 1.00 63.97 C ATOM 647 CD2 LEU A 127 221.38673.235 161.311 1.00 50.73 C ATOM 648 N TYR A 128 224.768 73.281 165.5881.00 85.19 N ATOM 649 CA TYR A 128 225.949 72.511 165.932 1.00 78.91 CATOM 650 C TYR A 128 225.657 71.015 166.036 1.00 99.93 C ATOM 651 O TYRA 128 224.960 70.571 166.956 1.00 83.06 O ATOM 652 CB TYR A 128 226.51372.996 167.259 1.00 73.34 C ATOM 653 CG TYR A 128 227.849 72.396 167.5621.00 113.80 C ATOM 654 CD1 TYR A 128 229.000 72.939 167.024 1.00 118.71C ATOM 655 CD2 TYR A 128 227.959 71.253 168.341 1.00 128.83 C ATOM 656CE1 TYR A 128 230.223 72.372 167.247 1.00 148.93 C ATOM 657 CE2 TYR A128 229.183 70.667 168.569 1.00 160.37 C ATOM 658 CZ TYR A 128 230.31671.239 168.018 1.00 162.93 C ATOM 659 OH TYR A 128 231.559 70.688168.244 1.00 162.00 O ATOM 660 N ALA A 129 226.204 70.240 165.098 1.00112.77 N ATOM 661 CA ALA A 129 226.016 68.788 165.077 1.00 119.60 C ATOM662 C ALA A 129 224.556 68.445 164.843 1.00 117.04 C ATOM 663 O ALA A129 223.981 67.637 165.575 1.00 102.48 O ATOM 664 CB ALA A 129 226.48268.181 166.398 1.00 105.12 C ATOM 665 N GLY A 130 223.957 69.056 163.8231.00 115.24 N ATOM 666 CA GLY A 130 222.550 68.810 163.553 1.00 115.49 CATOM 667 C GLY A 130 221.728 69.250 164.756 1.00 107.44 C ATOM 668 O GLYA 130 220.501 69.259 164.722 1.00 95.78 O ATOM 669 N ARG A 131 222.42569.616 165.828 1.00 89.82 N ATOM 670 CA ARG A 131 221.791 70.067 167.0551.00 92.75 C ATOM 671 C ARG A 131 221.754 71.593 167.105 1.00 80.22 CATOM 672 O ARG A 131 222.756 72.265 166.892 1.00 63.19 O ATOM 673 CB ARGA 131 222.546 69.516 168.271 1.00 79.11 C ATOM 674 N LYS A 132 220.57472.121 167.381 1.00 68.38 N ATOM 675 CA LYS A 132 220.349 73.553 167.4871.00 64.95 C ATOM 676 C LYS A 132 220.944 74.013 168.828 1.00 56.62 CATOM 677 O LYS A 132 220.449 73.648 169.889 1.00 63.01 O ATOM 678 CB LYSA 132 218.839 73.782 167.429 1.00 50.70 C ATOM 679 CG LYS A 132 218.34075.188 167.516 1.00 60.30 C ATOM 680 CD LYS A 132 216.821 75.123 167.4761.00 72.13 C ATOM 681 CE LYS A 132 216.171 76.486 167.371 1.00 106.61 CATOM 682 NZ LYS A 132 214.692 76.354 167.313 1.00 65.96 N ATOM 683 N CYSA 133 222.011 74.805 168.787 1.00 77.15 N ATOM 684 CA CYS A 133 222.64875.263 170.022 1.00 74.16 C ATOM 685 C CYS A 133 222.555 76.745 170.3201.00 65.05 C ATOM 686 O CYS A 133 222.157 77.543 169.471 1.00 81.10 OATOM 687 CB CYS A 133 224.114 74.877 170.024 1.00 92.23 C ATOM 688 SGCYS A 133 224.343 73.126 169.850 1.00 106.91 S ATOM 689 N LEU A 134222.947 77.099 171.540 1.00 67.21 N ATOM 690 CA LEU A 134 222.921 78.478171.986 1.00 67.45 C ATOM 691 C LEU A 134 224.326 78.865 172.431 1.0063.74 C ATOM 692 O LEU A 134 224.804 78.446 173.488 1.00 71.31 O ATOM693 CB LEU A 134 221.953 78.634 173.152 1.00 51.96 C ATOM 694 CG LEU A134 221.107 79.902 173.189 1.00 72.36 C ATOM 695 CD1 LEU A 134 220.12979.885 172.025 1.00 66.47 C ATOM 696 CD2 LEU A 134 220.342 79.981174.503 1.00 63.46 C ATOM 697 N LEU A 135 224.992 79.663 171.608 1.0069.76 N ATOM 698 CA LEU A 135 226.338 80.103 171.910 1.00 48.46 C ATOM699 C LEU A 135 226.262 81.450 172.608 1.00 60.20 C ATOM 700 O LEU A 135225.639 82.381 172.101 1.00 49.35 O ATOM 701 CB LEU A 135 227.130 80.192170.609 1.00 69.61 C ATOM 702 CG LEU A 135 227.191 78.851 169.873 1.0059.09 C ATOM 703 CD1 LEU A 135 227.585 79.058 168.425 1.00 80.07 C ATOM704 CD2 LEU A 135 228.173 77.943 170.579 1.00 92.22 C ATOM 705 N ILE A136 226.896 81.530 173.775 1.00 39.79 N ATOM 706 CA ILE A 136 226.91382.744 174.581 1.00 48.18 C ATOM 707 C ILE A 136 228.321 83.324 174.6911.00 54.20 C ATOM 708 O ILE A 136 229.219 82.672 175.204 1.00 63.12 OATOM 709 CB ILE A 136 226.398 82.453 176.006 1.00 57.17 C ATOM 710 CG1ILE A 136 224.997 81.864 175.934 1.00 54.12 C ATOM 711 CG2 ILE A 136226.364 83.720 176.829 1.00 45.37 C ATOM 712 CD1 ILE A 136 224.89380.475 176.545 1.00 94.13 C ATOM 713 N VAL A 137 228.527 84.539 174.2001.00 47.11 N ATOM 714 CA VAL A 137 229.812 85.185 174.376 1.00 51.92 CATOM 715 C VAL A 137 229.827 86.081 175.615 1.00 62.30 C ATOM 716 O VALA 137 228.925 86.871 175.875 1.00 65.38 O ATOM 717 CB VAL A 137 230.09185.979 173.099 1.00 55.29 C ATOM 718 CG1 VAL A 137 231.461 86.640173.162 1.00 49.20 C ATOM 719 CG2 VAL A 137 230.064 85.035 171.910 1.0033.49 C ATOM 720 N MET A 138 230.893 85.889 176.417 1.00 63.64 N ATOM721 CA MET A 138 231.026 86.598 177.685 1.00 47.48 C ATOM 722 C MET A138 232.339 87.381 177.769 1.00 63.41 C ATOM 723 O MET A 138 233.37186.975 177.246 1.00 60.31 O ATOM 724 CB MET A 138 231.044 85.548 178.7961.00 76.63 C ATOM 725 CG MET A 138 229.707 84.836 178.965 1.00 72.52 CATOM 726 SD MET A 138 229.779 83.570 180.236 1.00 81.89 S ATOM 727 CEMET A 138 228.109 82.937 180.029 1.00 180.26 C ATOM 728 N GLU A 139232.281 88.555 178.427 1.00 44.14 N ATOM 729 CA GLU A 139 233.550 89.154178.798 1.00 46.81 C ATOM 730 C GLU A 139 234.381 88.099 179.511 1.0061.98 C ATOM 731 O GLU A 139 233.876 87.285 180.272 1.00 68.61 O ATOM732 CB GLU A 139 233.291 90.346 179.732 1.00 40.20 C ATOM 733 CG GLU A139 233.444 89.999 181.216 1.00 70.67 C ATOM 734 CD GLU A 139 233.14091.231 182.059 1.00 76.30 C ATOM 735 OE1 GLU A 139 231.972 91.501182.306 1.00 89.15 O ATOM 736 OE2 GLU A 139 234.079 91.912 182.459 1.00100.67 O ATOM 737 N CYS A 140 235.664 88.142 179.172 1.00 69.16 N ATOM738 CA CYS A 140 236.625 87.167 179.666 1.00 69.69 C ATOM 739 C CYS A140 237.322 87.529 180.968 1.00 60.14 C ATOM 740 O CYS A 140 238.22788.349 180.990 1.00 82.48 O ATOM 741 CB CYS A 140 237.676 86.903 178.5871.00 74.24 C ATOM 742 SG CYS A 140 238.996 85.811 179.117 1.00 73.10 SATOM 743 N LEU A 141 236.912 86.895 182.055 1.00 71.80 N ATOM 744 CA LEUA 141 237.524 87.159 183.348 1.00 79.00 C ATOM 745 C LEU A 141 238.78586.304 183.509 1.00 76.59 C ATOM 746 O LEU A 141 238.736 85.081 183.3601.00 77.59 O ATOM 747 CB LEU A 141 236.523 86.847 184.462 1.00 81.97 CATOM 748 CG LEU A 141 235.203 87.610 184.350 1.00 76.77 C ATOM 749 CD1LEU A 141 234.214 87.100 185.382 1.00 96.50 C ATOM 750 CD2 LEU A 141235.469 89.086 184.535 1.00 47.85 C ATOM 751 N ASP A 142 239.913 86.940183.818 1.00 90.64 N ATOM 752 CA ASP A 142 241.158 86.197 183.977 1.0092.77 C ATOM 753 C ASP A 142 241.771 86.152 185.366 1.00 84.21 C ATOM754 O ASP A 142 242.387 85.153 185.725 1.00 120.09 O ATOM 755 CB ASP A142 242.202 86.698 182.984 1.00 92.82 C ATOM 756 CG ASP A 142 241.95186.182 181.586 1.00 110.94 C ATOM 757 OD1 ASP A 142 241.710 84.961181.450 1.00 82.18 O ATOM 758 OD2 ASP A 142 242.003 86.987 180.632 1.00115.53 O ATOM 759 N GLY A 143 241.616 87.220 186.142 1.00 76.67 N ATOM760 CA GLY A 143 242.178 87.249 187.486 1.00 84.99 C ATOM 761 C GLY A143 242.128 85.940 188.269 1.00 83.56 C ATOM 762 O GLY A 143 242.90985.744 189.194 1.00 94.38 O ATOM 763 N GLY A 144 241.216 85.040 187.9131.00 81.49 N ATOM 764 CA GLY A 144 241.132 83.778 188.621 1.00 72.81 CATOM 765 C GLY A 144 240.064 83.784 189.700 1.00 88.08 C ATOM 766 O GLYA 144 239.527 84.839 190.045 1.00 76.11 O ATOM 767 N GLU A 145 239.75982.603 190.235 1.00 72.60 N ATOM 768 CA GLU A 145 238.746 82.464 191.2681.00 80.92 C ATOM 769 C GLU A 145 239.050 83.249 192.539 1.00 85.24 CATOM 770 O GLU A 145 240.206 83.446 192.908 1.00 76.66 O ATOM 771 CB GLUA 145 238.543 80.993 191.605 1.00 61.62 C ATOM 772 CG GLU A 145 237.79780.215 190.539 1.00 89.57 C ATOM 773 CD GLU A 145 238.090 78.727 190.6181.00 126.57 C ATOM 774 OE1 GLU A 145 237.257 77.922 190.143 1.00 97.67 OATOM 775 OE2 GLU A 145 239.165 78.367 191.156 1.00 103.63 O ATOM 776 NLEU A 146 237.988 83.679 193.210 1.00 97.07 N ATOM 777 CA LEU A 146238.095 84.479 194.419 1.00 86.04 C ATOM 778 C LEU A 146 239.174 84.065195.402 1.00 75.32 C ATOM 779 O LEU A 146 240.049 84.852 195.732 1.0072.90 O ATOM 780 CB LEU A 146 236.743 84.530 195.138 1.00 83.71 C ATOM781 CG LEU A 146 236.768 85.402 196.395 1.00 84.94 C ATOM 782 CD1 LEU A146 237.338 86.754 196.044 1.00 92.90 C ATOM 783 CD2 LEU A 146 235.38285.551 196.975 1.00 116.24 C ATOM 784 N PHE A 147 239.127 82.829 195.8671.00 80.55 N ATOM 785 CA PHE A 147 240.104 82.385 196.847 1.00 87.51 CATOM 786 C PHE A 147 241.526 82.244 196.332 1.00 92.39 C ATOM 787 O PHEA 147 242.478 82.546 197.051 1.00 104.70 O ATOM 788 CB PHE A 147 239.61181.094 197.495 1.00 62.88 C ATOM 789 CG PHE A 147 238.388 81.297 198.3301.00 83.58 C ATOM 790 CD1 PHE A 147 237.514 80.260 198.589 1.00 80.71 CATOM 791 CD2 PHE A 147 238.102 82.557 198.845 1.00 82.88 C ATOM 792 CE1PHE A 147 236.375 80.476 199.342 1.00 93.95 C ATOM 793 CE2 PHE A 147236.971 82.778 199.596 1.00 50.19 C ATOM 794 CZ PHE A 147 236.105 81.740199.844 1.00 97.28 C ATOM 795 N SER A 148 241.680 81.805 195.090 1.0081.24 N ATOM 796 CA SER A 148 243.008 81.657 194.525 1.00 74.75 C ATOM797 C SER A 148 243.758 82.970 194.714 1.00 83.42 C ATOM 798 O SER A 148244.679 83.053 195.529 1.00 88.34 O ATOM 799 CB SER A 148 242.902 81.301193.051 1.00 76.50 C ATOM 800 OG SER A 148 242.158 80.106 192.902 1.0074.94 O ATOM 801 N ARG A 149 243.347 83.999 193.978 1.00 81.76 N ATOM802 CA ARG A 149 243.974 85.316 194.077 1.00 102.63 C ATOM 803 C ARG A149 244.325 85.650 195.512 1.00 107.57 C ATOM 804 O ARG A 149 245.39986.169 195.796 1.00 115.11 O ATOM 805 CB ARG A 149 243.038 86.396193.537 1.00 92.17 C ATOM 806 CG ARG A 149 242.950 86.427 192.033 1.0096.27 C ATOM 807 CD ARG A 149 244.278 86.851 191.433 1.00 127.89 C ATOM808 NE ARG A 149 244.141 88.079 190.656 1.00 105.03 N ATOM 809 CZ ARG A149 243.698 89.227 191.155 1.00 106.80 C ATOM 810 NH1 ARG A 149 243.35089.307 192.434 1.00 91.41 N ATOM 811 NH2 ARG A 149 243.597 90.293190.377 1.00 81.30 N ATOM 812 N ILE A 150 243.410 85.352 196.423 1.00117.64 N ATOM 813 CA ILE A 150 243.658 85.636 197.821 1.00 111.89 C ATOM814 C ILE A 150 244.890 84.907 198.320 1.00 111.27 C ATOM 815 O ILE A150 245.841 85.543 198.769 1.00 125.08 O ATOM 816 CB ILE A 150 242.44785.262 198.691 1.00 102.67 C ATOM 817 CG1 ILE A 150 241.501 86.458198.783 1.00 81.93 C ATOM 818 CG2 ILE A 150 242.900 84.866 200.081 1.0093.62 C ATOM 819 CD1 ILE A 150 241.279 87.159 197.457 1.00 131.92 C ATOM820 N GLN A 151 244.898 83.583 198.221 1.00 93.05 N ATOM 821 CA GLN A151 246.045 82.836 198.708 1.00 112.82 C ATOM 822 C GLN A 151 247.32583.240 197.992 1.00 130.96 C ATOM 823 O GLN A 151 248.373 83.378 198.6241.00 146.77 O ATOM 824 CB GLN A 151 245.825 81.329 198.565 1.00 96.24 CATOM 825 CG GLN A 151 246.123 80.751 197.203 1.00 107.91 C ATOM 826 CDGLN A 151 246.134 79.231 197.224 1.00 135.63 C ATOM 827 OE1 GLN A 151246.326 78.584 196.193 1.00 152.64 O ATOM 828 NE2 GLN A 151 245.92978.654 198.406 1.00 63.64 N ATOM 829 N ASP A 152 247.241 83.452 196.6821.00 131.86 N ATOM 830 CA ASP A 152 248.416 83.833 195.898 1.00 121.88 CATOM 831 C ASP A 152 248.697 85.331 195.974 1.00 119.03 C ATOM 832 O ASPA 152 249.617 85.765 196.678 1.00 108.04 O ATOM 833 CB ASP A 152 248.22683.420 194.436 1.00 120.82 C ATOM 834 CG ASP A 152 247.643 82.015194.296 1.00 145.66 C ATOM 835 OD1 ASP A 152 248.152 81.088 194.964 1.00161.64 O ATOM 836 OD2 ASP A 152 246.679 81.834 193.515 1.00 172.99 O TER836 ASP A 152 ATOM 837 N THR A 159 244.002 91.956 205.011 1.00 65.30 NATOM 838 CA THR A 159 243.414 92.956 205.898 1.00 99.51 C ATOM 839 C THRA 159 241.893 92.871 205.964 1.00 87.22 C ATOM 840 O THR A 159 241.22192.755 204.944 1.00 90.56 O ATOM 841 CB THR A 159 243.790 94.404 205.4731.00 95.19 C ATOM 842 OG1 THR A 159 243.610 94.560 204.061 1.00 103.02 OATOM 843 CG2 THR A 159 245.229 94.718 205.831 1.00 121.85 C ATOM 844 NGLU A 160 241.360 92.931 207.178 1.00 90.46 N ATOM 845 CA GLU A 160239.921 92.876 207.395 1.00 95.08 C ATOM 846 C GLU A 160 239.243 93.816206.400 1.00 97.10 C ATOM 847 O GLU A 160 238.191 93.498 205.844 1.0077.77 O ATOM 848 CB GLU A 160 239.606 93.302 208.836 1.00 84.05 C ATOM849 CG GLU A 160 238.135 93.255 209.218 1.00 102.30 C ATOM 850 CD GLU A160 237.888 93.636 210.675 1.00 107.68 C ATOM 851 OE1 GLU A 160 238.51293.022 211.566 1.00 102.89 O ATOM 852 OE2 GLU A 160 237.063 94.542210.931 1.00 116.03 O ATOM 853 N ARG A 161 239.872 94.966 206.169 1.0091.94 N ATOM 854 CA ARG A 161 239.359 95.979 205.252 1.00 98.89 C ATOM855 C ARG A 161 239.188 95.480 203.826 1.00 94.53 C ATOM 856 O ARG A 161238.251 95.874 203.129 1.00 84.43 O ATOM 857 CB ARG A 161 240.276 97.204205.245 1.00 94.61 C ATOM 858 CG ARG A 161 239.886 98.251 204.206 1.00116.99 C ATOM 859 CD ARG A 161 240.724 99.506 204.347 1.00 131.31 C ATOM860 NE ARG A 161 240.601 100.069 205.688 1.00 142.10 N ATOM 861 CZ ARG A161 241.205 101.180 206.092 1.00 132.03 C ATOM 862 NH1 ARG A 161 241.982101.855 205.257 1.00 142.54 N ATOM 863 NH2 ARG A 161 241.026 101.620207.330 1.00 138.86 N ATOM 864 N GLU A 162 240.107 94.628 203.389 1.0086.92 N ATOM 865 CA GLU A 162 240.040 94.067 202.050 1.00 96.77 C ATOM866 C GLU A 162 238.899 93.063 201.991 1.00 93.54 C ATOM 867 O GLU A 162238.101 93.078 201.052 1.00 98.52 O ATOM 868 CB GLU A 162 241.360 93.385201.699 1.00 101.33 C ATOM 869 CG GLU A 162 242.463 94.360 201.317 1.00125.60 C ATOM 870 CD GLU A 162 243.848 93.755 201.448 1.00 135.51 C ATOM871 OE1 GLU A 162 244.823 94.405 201.011 1.00 144.57 O ATOM 872 OE2 GLUA 162 243.959 92.636 201.999 1.00 130.61 O ATOM 873 N ALA A 163 238.82292.199 203.002 1.00 93.78 N ATOM 874 CA ALA A 163 237.772 91.187 203.0711.00 86.54 C ATOM 875 C ALA A 163 236.426 91.889 202.976 1.00 87.93 CATOM 876 O ALA A 163 235.509 91.418 202.297 1.00 94.67 O ATOM 877 CB ALAA 163 237.871 90.419 204.371 1.00 72.07 C ATOM 878 N SER A 164 236.31993.021 203.662 1.00 75.33 N ATOM 879 CA SER A 164 235.098 93.809 203.6511.00 81.45 C ATOM 880 C SER A 164 234.765 94.222 202.223 1.00 92.32 CATOM 881 O SER A 164 233.708 93.892 201.679 1.00 89.51 O ATOM 882 CB SERA 164 235.281 95.057 204.511 1.00 61.52 C ATOM 883 OG SER A 164 234.23395.977 204.289 1.00 85.01 O ATOM 884 N GLU A 165 235.696 94.949 201.6221.00 106.88 N ATOM 885 CA GLU A 165 235.537 95.436 200.259 1.00 107.60 CATOM 886 C GLU A 165 235.138 94.312 199.306 1.00 98.22 C ATOM 887 O GLUA 165 234.309 94.509 198.421 1.00 80.74 O ATOM 888 CB GLU A 165 236.83496.102 199.807 1.00 117.73 C ATOM 889 CG GLU A 165 237.296 97.198200.763 1.00 121.43 C ATOM 890 CD GLU A 165 238.640 97.776 200.381 1.00129.44 C ATOM 891 OE1 GLU A 165 239.620 96.998 200.285 1.00 132.54 OATOM 892 OE2 GLU A 165 238.711 99.007 200.172 1.00 114.59 O ATOM 893 NILE A 166 235.722 93.134 199.483 1.00 87.57 N ATOM 894 CA ILE A 166235.354 92.020 198.628 1.00 78.81 C ATOM 895 C ILE A 166 233.887 91.705198.905 1.00 85.13 C ATOM 896 O ILE A 166 233.068 91.644 197.975 1.0086.94 O ATOM 897 CB ILE A 166 236.207 90.755 198.905 1.00 76.17 C ATOM898 CG1 ILE A 166 237.628 90.972 198.376 1.00 76.65 C ATOM 899 CG2 ILE A166 235.567 89.534 198.255 1.00 77.07 C ATOM 900 CD1 ILE A 166 238.53289.759 198.515 1.00 56.87 C ATOM 901 N MET A 167 233.546 91.520 200.1791.00 80.82 N ATOM 902 CA MET A 167 232.166 91.195 200.521 1.00 77.70 CATOM 903 C MET A 167 231.228 92.224 199.941 1.00 73.89 C ATOM 904 O META 167 230.152 91.895 199.452 1.00 72.21 O ATOM 905 CB MET A 167 231.97891.110 202.035 1.00 67.42 C ATOM 906 CG MET A 167 232.544 89.845 202.6201.00 65.04 C ATOM 907 SD MET A 167 232.087 88.398 201.648 1.00 75.11 SATOM 908 CE MET A 167 230.294 88.399 201.807 1.00 53.41 C ATOM 909 N LYSA 168 231.659 93.472 199.962 1.00 54.97 N ATOM 910 CA LYS A 168 230.83094.534 199.449 1.00 60.76 C ATOM 911 C LYS A 168 230.456 94.386 197.9901.00 70.78 C ATOM 912 O LYS A 168 229.307 94.607 197.629 1.00 66.11 OATOM 913 CB LYS A 168 231.502 95.876 199.659 1.00 65.32 C ATOM 914 CGLYS A 168 230.616 97.027 199.293 1.00 49.49 C ATOM 915 CD LYS A 168231.208 98.313 199.780 1.00 103.77 C ATOM 916 CE LYS A 168 230.19199.429 199.714 1.00 87.12 C ATOM 917 NZ LYS A 168 230.644 100.674200.407 1.00 118.45 N ATOM 918 N SER A 169 231.403 94.023 197.136 1.0082.60 N ATOM 919 CA SER A 169 231.061 93.885 195.723 1.00 89.11 C ATOM920 C SER A 169 230.176 92.665 195.484 1.00 81.07 C ATOM 921 O SER A 169229.262 92.726 194.657 1.00 75.01 O ATOM 922 CB SER A 169 232.327 93.796194.863 1.00 73.74 C ATOM 923 OG SER A 169 233.058 92.620 195.165 1.0077.68 O ATOM 924 N ILE A 170 230.448 91.560 196.191 1.00 75.29 N ATOM925 CA ILE A 170 229.630 90.349 196.034 1.00 74.32 C ATOM 926 C ILE A170 228.232 90.743 196.437 1.00 63.33 C ATOM 927 O ILE A 170 227.25590.334 195.818 1.00 57.44 O ATOM 928 CB ILE A 170 230.053 89.177 196.9511.00 68.61 C ATOM 929 CG1 ILE A 170 231.437 88.663 196.571 1.00 59.95 CATOM 930 CG2 ILE A 170 229.091 88.015 196.782 1.00 46.17 C ATOM 931 CD1ILE A 170 231.768 87.318 197.192 1.00 76.19 C ATOM 932 N GLY A 171228.154 91.560 197.484 1.00 56.09 N ATOM 933 CA GLY A 171 226.870 92.026197.958 1.00 75.47 C ATOM 934 C GLY A 171 226.110 92.700 196.835 1.0069.45 C ATOM 935 O GLY A 171 224.947 92.390 196.568 1.00 76.12 O ATOM936 N GLU A 172 226.792 93.621 196.166 1.00 71.58 N ATOM 937 CA GLU A172 226.212 94.368 195.066 1.00 73.09 C ATOM 938 C GLU A 172 225.71593.446 193.960 1.00 69.34 C ATOM 939 O GLU A 172 224.645 93.653 193.3961.00 74.21 O ATOM 940 CB GLU A 172 227.243 95.332 194.499 1.00 62.80 CATOM 941 CG GLU A 172 228.028 96.080 195.547 1.00 91.94 C ATOM 942 CDGLU A 172 228.717 97.298 194.973 1.00 112.10 C ATOM 943 OE1 GLU A 172228.051 98.349 194.825 1.00 116.39 O ATOM 944 OE2 GLU A 172 229.91997.199 194.649 1.00 121.47 O ATOM 945 N ALA A 173 226.487 92.427 193.6351.00 50.91 N ATOM 946 CA ALA A 173 226.039 91.525 192.599 1.00 55.42 CATOM 947 C ALA A 173 224.666 90.969 192.995 1.00 69.00 C ATOM 948 O ALAA 173 223.779 90.793 192.156 1.00 68.62 O ATOM 949 CB ALA A 173 227.03690.392 192.429 1.00 59.45 C ATOM 950 N ILE A 174 224.491 90.709 194.2871.00 59.68 N ATOM 951 CA ILE A 174 223.240 90.163 194.793 1.00 55.23 CATOM 952 C ILE A 174 222.137 91.199 194.912 1.00 66.27 C ATOM 953 O ILEA 174 220.991 90.943 194.542 1.00 59.24 O ATOM 954 CB ILE A 174 223.43989.528 196.157 1.00 72.24 C ATOM 955 CG1 ILE A 174 224.492 88.438196.055 1.00 44.08 C ATOM 956 CG2 ILE A 174 222.132 88.924 196.636 1.0052.34 C ATOM 957 CD1 ILE A 174 224.090 87.355 195.102 1.00 105.89 C ATOM958 N GLN A 175 222.473 92.368 195.444 1.00 46.22 N ATOM 959 CA GLN A175 221.477 93.407 195.586 1.00 60.91 C ATOM 960 C GLN A 175 220.80493.676 194.240 1.00 48.25 C ATOM 961 O GLN A 175 219.581 93.742 194.1551.00 62.61 O ATOM 962 CB GLN A 175 222.121 94.674 196.144 1.00 49.40 CATOM 963 CG GLN A 175 221.309 95.931 195.912 1.00 71.12 C ATOM 964 CDGLN A 175 221.661 97.042 196.892 1.00 104.15 C ATOM 965 OE1 GLN A 175222.825 97.446 197.017 1.00 76.77 O ATOM 966 NE2 GLN A 175 220.64597.544 197.600 1.00 111.29 N ATOM 967 N TYR A 176 221.602 93.798 193.1841.00 72.76 N ATOM 968 CA TYR A 176 221.025 94.078 191.873 1.00 74.81 CATOM 969 C TYR A 176 220.105 92.946 191.416 1.00 74.26 C ATOM 970 O TYRA 176 218.933 93.142 191.122 1.00 61.26 O ATOM 971 CB TYR A 176 222.16894.255 190.874 1.00 79.19 C ATOM 972 CG TYR A 176 221.628 94.703 189.5631.00 53.89 C ATOM 973 CD1 TYR A 176 221.692 96.048 189.209 1.00 73.14 CATOM 974 CD2 TYR A 176 221.054 93.785 188.683 1.00 78.08 C ATOM 975 CE1TYR A 176 221.197 96.471 187.985 1.00 58.55 C ATOM 976 CE2 TYR A 176220.550 94.209 187.463 1.00 60.19 C ATOM 977 CZ TYR A 176 220.616 95.546187.115 1.00 70.05 C ATOM 978 OH TYR A 176 220.124 95.975 185.898 1.0098.35 O ATOM 979 N LEU A 177 220.626 91.729 191.357 1.00 69.60 N ATOM980 CA LEU A 177 219.824 90.582 190.986 1.00 65.69 C ATOM 981 C LEU A177 218.485 90.616 191.705 1.00 62.46 C ATOM 982 O LEU A 177 217.43790.563 191.057 1.00 73.48 O ATOM 983 CB LEU A 177 220.562 89.297 191.3331.00 59.94 C ATOM 984 CG LEU A 177 221.671 88.955 190.357 1.00 54.02 CATOM 985 CD1 LEU A 177 222.252 87.585 190.679 1.00 59.89 C ATOM 986 CD2LEU A 177 221.078 88.962 188.964 1.00 77.33 C ATOM 987 N HIS A 178218.518 90.708 193.035 1.00 61.49 N ATOM 988 CA HIS A 178 217.285 90.746193.816 1.00 57.69 C ATOM 989 C HIS A 178 216.419 91.952 193.466 1.0056.16 C ATOM 990 O HIS A 178 215.204 91.831 193.368 1.00 71.55 O ATOM991 CB HIS A 178 217.590 90.741 195.313 1.00 58.67 C ATOM 992 CG HIS A178 218.154 89.446 195.805 1.00 66.35 C ATOM 993 ND1 HIS A 178 218.55489.259 197.110 1.00 49.65 N ATOM 994 CD2 HIS A 178 218.437 88.292195.154 1.00 50.67 C ATOM 995 CE1 HIS A 178 219.068 88.049 197.241 1.0077.64 C ATOM 996 NE2 HIS A 178 219.010 87.442 196.069 1.00 44.53 N ATOM997 N SER A 179 217.037 93.107 193.259 1.00 62.56 N ATOM 998 CA SER A179 216.270 94.292 192.906 1.00 56.93 C ATOM 999 C SER A 179 215.57594.094 191.567 1.00 44.23 C ATOM 1000 O SER A 179 214.830 94.956 191.1361.00 68.45 O ATOM 1001 CB SER A 179 217.160 95.518 192.803 1.00 41.33 CATOM 1002 OG SER A 179 217.835 95.504 191.554 1.00 86.61 O ATOM 1003 NILE A 180 215.842 92.997 190.876 1.00 60.70 N ATOM 1004 CA ILE A 180215.135 92.778 189.629 1.00 66.56 C ATOM 1005 C ILE A 180 214.483 91.409189.667 1.00 76.38 C ATOM 1006 O ILE A 180 214.223 90.783 188.641 1.0067.28 O ATOM 1007 CB ILE A 180 216.041 92.928 188.368 1.00 65.72 C ATOM1008 CG1 ILE A 180 217.061 91.804 188.280 1.00 98.00 C ATOM 1009 CG2 ILEA 180 216.766 94.248 188.413 1.00 65.05 C ATOM 1010 CD1 ILE A 180217.787 91.783 186.962 1.00 42.99 C ATOM 1011 N ASN A 181 214.229 90.940190.880 1.00 62.76 N ATOM 1012 CA ASN A 181 213.556 89.672 191.081 1.0064.37 C ATOM 1013 C ASN A 181 214.257 88.447 190.552 1.00 64.72 C ATOM1014 O ASN A 181 213.624 87.605 189.919 1.00 76.26 O ATOM 1015 CB ASN A181 212.156 89.726 190.464 1.00 52.97 C ATOM 1016 CG ASN A 181 211.27490.765 191.118 1.00 91.87 C ATOM 1017 OD1 ASN A 181 210.607 91.549190.435 1.00 70.51 O ATOM 1018 ND2 ASN A 181 211.261 90.779 192.456 1.0066.84 N ATOM 1019 N ILE A 182 215.550 88.325 190.800 1.00 69.03 N ATOM1020 CA ILE A 182 216.251 87.140 190.343 1.00 58.61 C ATOM 1021 C ILE A182 217.060 86.582 191.510 1.00 64.49 C ATOM 1022 O ILE A 182 217.65287.335 192.289 1.00 75.24 O ATOM 1023 CB ILE A 182 217.217 87.452189.175 1.00 67.77 C ATOM 1024 CG1 ILE A 182 216.498 88.204 188.058 1.0051.81 C ATOM 1025 CG2 ILE A 182 217.765 86.162 188.604 1.00 71.00 C ATOM1026 CD1 ILE A 182 217.397 88.520 186.870 1.00 51.04 C ATOM 1027 N ALA A183 217.068 85.261 191.639 1.00 74.62 N ATOM 1028 CA ALA A 183 217.90784.570 192.612 1.00 86.67 C ATOM 1029 C ALA A 183 218.970 83.710 191.9241.00 88.25 C ATOM 1030 O ALA A 183 218.683 82.841 191.110 1.00 87.52 OATOM 1031 CB ALA A 183 217.006 83.691 193.480 1.00 82.75 C ATOM 1032 NHIS A 184 220.241 84.008 192.248 1.00 80.72 N ATOM 1033 CA HIS A 184221.331 83.245 191.654 1.00 76.94 C ATOM 1034 C HIS A 184 221.282 81.775192.077 1.00 80.75 C ATOM 1035 O HIS A 184 221.411 80.858 191.277 1.0072.26 O ATOM 1036 CB HIS A 184 222.652 83.872 192.102 1.00 71.67 C ATOM1037 CG HIS A 184 223.777 83.319 191.264 1.00 61.47 C ATOM 1038 ND1 HISA 184 224.599 84.095 190.515 1.00 83.49 N ATOM 1039 CD2 HIS A 184224.158 81.983 191.101 1.00 84.93 C ATOM 1040 CE1 HIS A 184 225.45683.245 189.918 1.00 77.60 C ATOM 1041 NE2 HIS A 184 225.215 81.974190.250 1.00 94.64 N ATOM 1042 N ARG A 185 221.128 81.570 193.401 1.0076.53 N ATOM 1043 CA ARG A 185 221.070 80.207 193.915 1.00 77.64 C ATOM1044 C ARG A 185 222.301 79.398 193.501 1.00 85.59 C ATOM 1045 O ARG A185 222.247 78.190 193.308 1.00 76.00 O ATOM 1046 CB ARG A 185 219.80579.546 193.368 1.00 60.05 C ATOM 1047 CG ARG A 185 218.538 80.313193.747 1.00 69.87 C ATOM 1048 CD ARG A 185 217.293 79.421 193.714 1.0088.79 C ATOM 1049 NE ARG A 185 216.984 79.018 192.339 1.00 74.62 N ATOM1050 CZ ARG A 185 216.704 77.720 192.125 1.00 102.74 C ATOM 1051 NH1 ARGA 185 216.704 76.865 193.132 1.00 105.16 N ATOM 1052 NH2 ARG A 185216.428 77.299 190.884 1.00 124.15 N ATOM 1053 N ASP A 186 223.47680.000 193.397 1.00 85.17 N ATOM 1054 CA ASP A 186 224.677 79.238193.128 1.00 65.94 C ATOM 1055 C ASP A 186 225.857 80.161 193.327 1.0063.69 C ATOM 1056 O ASP A 186 226.821 80.122 192.573 1.00 76.80 O ATOM1057 CB ASP A 186 224.651 78.670 191.712 1.00 64.22 C ATOM 1058 CG ASP A186 225.560 77.461 191.555 1.00 69.83 C ATOM 1059 OD1 ASP A 186 225.62976.654 192.503 1.00 114.36 O ATOM 1060 OD2 ASP A 186 226.197 77.304190.492 1.00 103.36 O ATOM 1061 N VAL A 187 225.757 80.991 194.364 1.0065.08 N ATOM 1062 CA VAL A 187 226.794 81.952 194.717 1.00 62.87 C ATOM1063 C VAL A 187 227.923 81.305 195.507 1.00 61.46 C ATOM 1064 O VAL A187 228.134 81.605 196.677 1.00 76.23 O ATOM 1065 CB VAL A 187 226.21883.132 195.539 1.00 56.36 C ATOM 1066 CG1 VAL A 187 227.311 84.156195.831 1.00 45.04 C ATOM 1067 CG2 VAL A 187 225.099 83.791 194.774 1.0049.15 C ATOM 1068 N LYS A 188 228.644 80.407 194.852 1.00 71.26 N ATOM1069 CA LYS A 188 229.766 79.731 195.482 1.00 71.74 C ATOM 1070 C LYS A188 231.033 80.392 194.985 1.00 71.11 C ATOM 1071 O LYS A 188 231.04980.975 193.905 1.00 76.85 O ATOM 1072 CB LYS A 188 229.770 78.246195.123 1.00 59.58 C ATOM 1073 CG LYS A 188 229.709 77.962 193.639 1.0061.04 C ATOM 1074 CD LYS A 188 228.939 76.679 193.367 1.00 96.00 C ATOM1075 CE LYS A 188 228.669 76.509 191.869 1.00 141.45 C ATOM 1076 NZ LYSA 188 227.746 75.371 191.579 1.00 122.86 N ATOM 1077 N PRO A 189 232.11580.318 195.776 1.00 66.86 N ATOM 1078 CA PRO A 189 233.409 80.911195.433 1.00 82.94 C ATOM 1079 C PRO A 189 233.746 80.698 193.967 1.0081.19 C ATOM 1080 O PRO A 189 233.989 81.641 193.231 1.00 92.51 O ATOM1081 CB PRO A 189 234.359 80.199 196.381 1.00 77.38 C ATOM 1082 CG PRO A189 233.511 80.063 197.620 1.00 79.38 C ATOM 1083 CD PRO A 189 232.19779.582 197.049 1.00 84.38 C ATOM 1084 N GLU A 190 233.743 79.447 193.5531.00 79.12 N ATOM 1085 CA GLU A 190 234.019 79.080 192.178 1.00 82.41 CATOM 1086 C GLU A 190 233.441 80.092 191.159 1.00 81.32 C ATOM 1087 OGLU A 190 234.121 80.487 190.213 1.00 102.09 O ATOM 1088 CB GLU A 190233.432 77.689 191.920 1.00 94.71 C ATOM 1089 CG GLU A 190 233.78476.609 192.978 1.00 121.01 C ATOM 1090 CD GLU A 190 232.689 76.364194.043 1.00 135.59 C ATOM 1091 OE1 GLU A 190 231.485 76.334 193.6931.00 113.86 O ATOM 1092 OE2 GLU A 190 233.039 76.185 195.236 1.00 133.59O ATOM 1093 N ASN A 191 232.196 80.527 191.350 1.00 80.04 N ATOM 1094 CAASN A 191 231.584 81.457 190.405 1.00 59.50 C ATOM 1095 C ASN A 191231.991 82.905 190.569 1.00 72.63 C ATOM 1096 O ASN A 191 231.360 83.796189.998 1.00 60.72 O ATOM 1097 CB ASN A 191 230.068 81.364 190.471 1.0059.13 C ATOM 1098 CG ASN A 191 229.566 79.975 190.189 1.00 57.02 C ATOM1099 OD1 ASN A 191 228.797 79.413 190.959 1.00 107.48 O ATOM 1100 ND2ASN A 191 230.005 79.406 189.077 1.00 62.85 N ATOM 1101 N LEU A 192233.042 83.149 191.342 1.00 64.74 N ATOM 1102 CA LEU A 192 233.52484.514 191.552 1.00 58.75 C ATOM 1103 C LEU A 192 234.931 84.655 190.9871.00 67.25 C ATOM 1104 O LEU A 192 235.906 84.314 191.644 1.00 63.30 OATOM 1105 CB LEU A 192 233.512 84.856 193.047 1.00 64.92 C ATOM 1106 CGLEU A 192 232.127 84.840 193.709 1.00 76.84 C ATOM 1107 CD1 LEU A 192232.285 84.706 195.207 1.00 63.79 C ATOM 1108 CD2 LEU A 192 231.35086.108 193.349 1.00 49.09 C ATOM 1109 N LEU A 193 235.022 85.173 189.7671.00 76.60 N ATOM 1110 CA LEU A 193 236.299 85.334 189.077 1.00 68.12 CATOM 1111 C LEU A 193 236.765 86.779 189.008 1.00 67.18 C ATOM 1112 OLEU A 193 235.957 87.700 189.001 1.00 70.12 O ATOM 1113 CB LEU A 193236.165 84.801 187.657 1.00 57.38 C ATOM 1114 CG LEU A 193 235.34683.517 187.573 1.00 67.03 C ATOM 1115 CD1 LEU A 193 235.242 83.061186.137 1.00 74.99 C ATOM 1116 CD2 LEU A 193 235.985 82.449 188.445 1.0080.12 C ATOM 1117 N TYR A 194 238.073 86.982 188.950 1.00 66.38 N ATOM1118 CA TYR A 194 238.603 88.333 188.846 1.00 70.20 C ATOM 1119 C TYR A194 238.833 88.697 187.382 1.00 65.05 C ATOM 1120 O TYR A 194 239.21987.856 186.578 1.00 55.59 O ATOM 1121 CB TYR A 194 239.903 88.449189.636 1.00 71.53 C ATOM 1122 CG TYR A 194 239.692 88.807 191.085 1.0083.55 C ATOM 1123 CD1 TYR A 194 239.221 90.068 191.446 1.00 60.55 C ATOM1124 CD2 TYR A 194 239.959 87.889 192.095 1.00 54.69 C ATOM 1125 CE1 TYRA 194 239.026 90.402 192.766 1.00 77.54 C ATOM 1126 CE2 TYR A 194239.764 88.216 193.423 1.00 72.23 C ATOM 1127 CZ TYR A 194 239.30089.472 193.749 1.00 80.69 C ATOM 1128 OH TYR A 194 239.118 89.812195.068 1.00 97.12 O ATOM 1129 N THR A 195 238.585 89.952 187.035 1.0056.71 N ATOM 1130 CA THR A 195 238.754 90.389 185.660 1.00 63.48 C ATOM1131 C THR A 195 240.193 90.206 185.176 1.00 82.40 C ATOM 1132 O THR A195 240.436 89.542 184.164 1.00 96.10 O ATOM 1133 CB THR A 195 238.33491.865 185.494 1.00 66.82 C ATOM 1134 OG1 THR A 195 239.028 92.668186.452 1.00 70.80 O ATOM 1135 CG2 THR A 195 236.829 92.022 185.688 1.0054.17 C ATOM 1136 N SER A 196 241.145 90.790 185.897 1.00 84.27 N ATOM1137 CA SER A 196 242.552 90.672 185.532 1.00 77.62 C ATOM 1138 C SER A196 243.388 90.202 186.716 1.00 88.08 C ATOM 1139 O SER A 196 242.86589.980 187.805 1.00 101.07 O ATOM 1140 CB SER A 196 243.089 92.019185.033 1.00 78.67 C ATOM 1141 OG SER A 196 243.063 92.995 186.055 1.0092.46 O ATOM 1142 N LYS A 197 244.688 90.038 186.490 1.00 106.23 N ATOM1143 CA LYS A 197 245.601 89.609 187.542 1.00 100.72 C ATOM 1144 C LYS A197 246.055 90.885 188.230 1.00 93.19 C ATOM 1145 O LYS A 197 246.50990.870 189.373 1.00 91.43 O ATOM 1146 CB LYS A 197 246.792 88.882186.936 1.00 90.18 C ATOM 1147 N ARG A 198 245.911 91.985 187.500 1.0076.02 N ATOM 1148 CA ARG A 198 246.275 93.318 187.959 1.00 111.08 C ATOM1149 C ARG A 198 245.815 93.469 189.412 1.00 105.33 C ATOM 1150 O ARG A198 244.993 92.696 189.885 1.00 104.59 O ATOM 1151 CB ARG A 198 245.58294.345 187.048 1.00 118.16 C ATOM 1152 CG ARG A 198 246.321 95.666186.798 1.00 144.07 C ATOM 1153 CD ARG A 198 245.911 96.211 185.435 1.00152.31 C ATOM 1154 NE ARG A 198 246.262 97.611 185.210 1.00 171.50 NATOM 1155 CZ ARG A 198 245.920 98.290 184.115 1.00 162.32 C ATOM 1156NH1 ARG A 198 245.223 97.692 183.152 1.00 96.38 N ATOM 1157 NH2 ARG A198 246.265 99.565 183.984 1.00 146.82 N ATOM 1158 N PRO A 199 246.34794.461 190.144 1.00 115.43 N ATOM 1159 CA PRO A 199 245.943 94.653191.545 1.00 123.33 C ATOM 1160 C PRO A 199 244.581 95.315 191.719 1.00123.60 C ATOM 1161 O PRO A 199 243.991 95.259 192.794 1.00 135.30 O ATOM1162 CB PRO A 199 247.061 95.524 192.105 1.00 121.70 C ATOM 1163 CG PROA 199 247.387 96.397 190.921 1.00 133.24 C ATOM 1164 CD PRO A 199247.411 95.409 189.775 1.00 125.16 C ATOM 1165 N ASN A 200 244.09095.943 190.656 1.00 122.57 N ATOM 1166 CA ASN A 200 242.810 96.629190.719 1.00 108.91 C ATOM 1167 C ASN A 200 241.726 95.826 190.023 1.0095.83 C ATOM 1168 O ASN A 200 240.606 96.310 189.835 1.00 73.96 O ATOM1169 CB ASN A 200 242.923 98.019 190.090 1.00 118.71 C ATOM 1170 N ALA A201 242.059 94.598 189.637 1.00 75.18 N ATOM 1171 CA ALA A 201 241.09393.725 188.979 1.00 84.90 C ATOM 1172 C ALA A 201 239.789 93.860 189.7511.00 91.91 C ATOM 1173 O ALA A 201 239.800 94.214 190.929 1.00 89.51 OATOM 1174 CB ALA A 201 241.579 92.278 189.022 1.00 81.83 C ATOM 1175 NILE A 202 238.660 93.607 189.097 1.00 78.80 N ATOM 1176 CA ILE A 202237.378 93.698 189.795 1.00 91.70 C ATOM 1177 C ILE A 202 236.697 92.326189.886 1.00 95.28 C ATOM 1178 O ILE A 202 236.837 91.488 188.995 1.0088.21 O ATOM 1179 CB ILE A 202 236.454 94.707 189.121 1.00 76.30 C ATOM1180 CG1 ILE A 202 235.623 94.029 188.059 1.00 78.45 C ATOM 1181 CG2 ILEA 202 237.278 95.772 188.468 1.00 61.72 C ATOM 1182 CD1 ILE A 202234.681 94.991 187.390 1.00 169.28 C ATOM 1183 N LEU A 203 235.97592.102 190.981 1.00 93.25 N ATOM 1184 CA LEU A 203 235.308 90.821191.231 1.00 66.28 C ATOM 1185 C LEU A 203 233.935 90.746 190.587 1.0067.61 C ATOM 1186 O LEU A 203 233.137 91.681 190.695 1.00 68.70 O ATOM1187 CB LEU A 203 235.195 90.599 192.745 1.00 73.90 C ATOM 1188 CG LEU A203 234.947 89.189 193.273 1.00 52.16 C ATOM 1189 CD1 LEU A 203 236.01688.244 192.783 1.00 61.94 C ATOM 1190 CD2 LEU A 203 234.956 89.228194.779 1.00 85.31 C ATOM 1191 N LYS A 204 233.665 89.628 189.919 1.0058.34 N ATOM 1192 CA LYS A 204 232.392 89.434 189.243 1.00 58.89 C ATOM1193 C LYS A 204 231.788 88.047 189.423 1.00 73.88 C ATOM 1194 O LYS A204 232.490 87.027 189.406 1.00 67.59 O ATOM 1195 CB LYS A 204 232.54089.740 187.750 1.00 68.02 C ATOM 1196 CG LYS A 204 232.844 91.199187.461 1.00 64.40 C ATOM 1197 CD LYS A 204 232.603 91.552 186.008 1.0062.63 C ATOM 1198 CE LYS A 204 232.786 93.033 185.788 1.00 71.16 C ATOM1199 NZ LYS A 204 232.247 93.468 184.487 1.00 94.63 N ATOM 1200 N LEU A205 230.472 88.023 189.586 1.00 66.05 N ATOM 1201 CA LEU A 205 229.75086.781 189.782 1.00 63.28 C ATOM 1202 C LEU A 205 229.296 86.241 188.4521.00 53.73 C ATOM 1203 O LEU A 205 228.884 87.001 187.583 1.00 55.72 OATOM 1204 CB LEU A 205 228.540 87.024 190.680 1.00 64.69 C ATOM 1205 CGLEU A 205 227.514 85.905 190.813 1.00 47.19 C ATOM 1206 CD1 LEU A 205228.150 84.657 191.394 1.00 81.96 C ATOM 1207 CD2 LEU A 205 226.39786.387 191.695 1.00 64.88 C ATOM 1208 N THR A 206 229.374 84.926 188.3001.00 53.81 N ATOM 1209 CA THR A 206 228.976 84.363 187.015 1.00 65.26 CATOM 1210 C THR A 206 228.239 83.033 187.185 1.00 53.82 C ATOM 1211 OTHR A 206 228.098 82.495 188.276 1.00 71.37 O ATOM 1212 CB THR A 206230.236 84.153 186.174 1.00 61.13 C ATOM 1213 OG1 THR A 206 231.02383.115 186.762 1.00 57.86 O ATOM 1214 CG2 THR A 206 231.064 85.441186.137 1.00 74.17 C ATOM 1215 N ASP A 207 227.721 82.523 186.051 1.0079.42 N ATOM 1216 CA ASP A 207 227.033 81.239 186.082 1.00 59.92 C ATOM1217 C ASP A 207 225.577 81.381 186.532 1.00 58.53 C ATOM 1218 O ASP A207 225.272 81.747 187.660 1.00 80.39 O ATOM 1219 CB ASP A 207 227.78580.312 187.037 1.00 60.66 C ATOM 1220 CG ASP A 207 227.400 78.869186.743 1.00 80.61 C ATOM 1221 OD1 ASP A 207 226.540 78.671 185.884 1.0083.40 O ATOM 1222 OD2 ASP A 207 227.955 77.969 187.369 1.00 77.29 O ATOM1223 N PHE A 208 224.661 81.117 185.582 1.00 57.58 N ATOM 1224 CA PHE A208 223.242 81.177 185.911 1.00 65.42 C ATOM 1225 C PHE A 208 222.55479.836 185.650 1.00 55.77 C ATOM 1226 O PHE A 208 221.342 79.748 185.5041.00 71.33 O ATOM 1227 CB PHE A 208 222.597 82.267 185.055 1.00 41.42 CATOM 1228 CG PHE A 208 222.872 83.615 185.655 1.00 55.64 C ATOM 1229 CD1PHE A 208 224.177 84.088 185.707 1.00 30.42 C ATOM 1230 CD2 PHE A 208221.825 84.390 186.125 1.00 49.87 C ATOM 1231 CE1 PHE A 208 224.43285.349 186.229 1.00 68.91 C ATOM 1232 CE2 PHE A 208 222.089 85.654186.645 1.00 47.56 C ATOM 1233 CZ PHE A 208 223.390 86.139 186.697 1.0056.42 C ATOM 1234 N GLY A 209 223.362 78.788 185.594 1.00 61.07 N ATOM1235 CA GLY A 209 222.825 77.461 185.343 1.00 72.68 C ATOM 1236 C GLY A209 221.723 77.080 186.309 1.00 73.62 C ATOM 1237 O GLY A 209 220.95876.161 186.039 1.00 77.98 O ATOM 1238 N PHE A 210 221.656 77.777 187.4411.00 83.69 N ATOM 1239 CA PHE A 210 220.624 77.527 188.439 1.00 69.57 CATOM 1240 C PHE A 210 219.818 78.787 188.732 1.00 81.37 C ATOM 1241 OPHE A 210 218.872 78.738 189.507 1.00 58.29 O ATOM 1242 CB PHE A 210221.225 77.037 189.753 1.00 67.31 C ATOM 1243 CG PHE A 210 222.00475.774 189.633 1.00 73.06 C ATOM 1244 CD1 PHE A 210 221.429 74.645189.085 1.00 114.78 C ATOM 1245 CD2 PHE A 210 223.306 75.703 190.0971.00 90.32 C ATOM 1246 CE1 PHE A 210 222.134 73.459 189.000 1.00 109.98C ATOM 1247 CE2 PHE A 210 224.021 74.522 190.017 1.00 108.27 C ATOM 1248CZ PHE A 210 223.432 73.398 189.467 1.00 132.84 C ATOM 1249 N ALA A 211220.208 79.913 188.137 1.00 81.89 N ATOM 1250 CA ALA A 211 219.49081.170 188.341 1.00 69.02 C ATOM 1251 C ALA A 211 218.028 80.898 188.0751.00 58.89 C ATOM 1252 O ALA A 211 217.700 80.102 187.206 1.00 66.98 OATOM 1253 CB ALA A 211 219.992 82.228 187.381 1.00 73.43 C ATOM 1254 NLYS A 212 217.147 81.552 188.817 1.00 66.85 N ATOM 1255 CA LYS A 212215.726 81.329 188.624 1.00 74.67 C ATOM 1256 C LYS A 212 214.892 82.598188.785 1.00 69.94 C ATOM 1257 O LYS A 212 215.106 83.387 189.709 1.0068.45 O ATOM 1258 CB LYS A 212 215.242 80.249 189.600 1.00 70.15 C ATOM1259 CG LYS A 212 213.744 80.022 189.558 1.00 104.51 C ATOM 1260 CD LYSA 212 213.371 78.669 188.952 1.00 157.53 C ATOM 1261 CE LYS A 212211.877 78.354 189.164 1.00 119.37 C ATOM 1262 NZ LYS A 212 211.40877.209 188.336 1.00 93.54 N ATOM 1263 N GLU A 213 214.141 82.794 187.8681.00 15.00 ATOM 1264 CA GLU A 213 213.459 84.043 187.550 1.00 15.00 ATOM1265 CB GLU A 213 213.339 84.210 186.033 1.00 15.00 ATOM 1266 CG GLU A213 212.159 83.475 185.419 1.00 15.00 ATOM 1267 CD GLU A 213 212.04383.702 183.925 1.00 15.00 ATOM 1268 OE1 GLU A 213 211.180 83.058 183.2891.00 15.00 ATOM 1269 OE2 GLU A 213 212.815 84.522 183.385 1.00 15.00ATOM 1270 C GLU A 213 212.074 84.087 188.185 1.00 15.00 ATOM 1271 O GLUA 213 211.548 83.061 188.604 1.00 15.36 ATOM 1272 N THR A 214 211.49185.288 188.256 1.00 15.00 ATOM 1273 CA THR A 214 210.119 85.515 188.6931.00 15.00 ATOM 1274 CB THR A 214 209.149 84.520 188.029 1.00 15.00 ATOM1275 OG1 THR A 214 209.187 84.691 186.606 1.00 15.00 ATOM 1276 CG2 THR A214 207.729 84.751 188.525 1.00 15.00 ATOM 1277 C THR A 214 209.99985.380 190.208 1.00 15.00 ATOM 1278 O THR A 214 208.821 85.220 190.6841.00 20.56 ATOM 1279 N THR A 215 211.054 85.475 190.986 1.00 15.00 ATOM1280 CA THR A 215 210.986 85.364 192.438 1.00 15.00 ATOM 1281 CB THR A215 212.132 84.493 192.987 1.00 15.00 ATOM 1282 OG1 THR A 215 212.03183.170 192.444 1.00 15.00 ATOM 1283 CG2 THR A 215 212.062 84.420 194.5051.00 15.00 ATOM 1284 C THR A 215 211.057 86.739 193.095 1.00 15.00 ATOM1285 O THR A 215 212.111 87.316 193.358 1.00 17.54 ATOM 1286 N SER A 216209.845 87.327 193.310 1.00 15.00 ATOM 1287 CA SER A 216 209.645 88.633193.871 1.00 15.00 ATOM 1288 CB SER A 216 208.582 89.393 193.087 1.0015.00 ATOM 1289 OG SER A 216 207.344 88.725 193.050 1.00 15.00 ATOM 1290C SER A 216 209.352 88.649 195.380 1.00 15.00 ATOM 1291 O SER A 216209.553 89.753 195.944 1.00 24.37 ATOM 1292 N HIS A 217 208.923 87.546195.889 1.00 15.00 ATOM 1293 CA HIS A 217 208.587 87.117 197.213 1.0015.00 ATOM 1294 CB HIS A 217 208.248 88.231 198.187 1.00 15.00 ATOM 1295CG HIS A 217 208.489 87.994 199.627 1.00 15.00 ATOM 1296 CD2 HIS A 217209.559 88.383 200.390 1.00 15.00 ATOM 1297 ND1 HIS A 217 207.605 87.415200.500 1.00 15.00 ATOM 1298 CE1 HIS A 217 208.130 87.392 201.715 1.0015.00 ATOM 1299 NE2 HIS A 217 209.316 87.977 201.671 1.00 15.00 ATOM1300 C HIS A 217 207.430 86.096 197.115 1.00 15.00 ATOM 1301 O HIS A 217206.272 86.389 196.895 1.00 28.08 ATOM 1302 N ASN A 218 207.933 84.916197.149 1.00 15.00 ATOM 1303 CA ASN A 218 208.098 83.782 196.497 1.0015.00 ATOM 1304 CB ASN A 218 207.843 83.860 194.969 1.00 15.00 ATOM 1305CG ASN A 218 206.412 83.605 194.590 1.00 15.00 ATOM 1306 OD1 ASN A 218205.835 82.541 194.841 1.00 15.00 ATOM 1307 ND2 ASN A 218 205.794 84.575193.913 1.00 15.00 ATOM 1308 C ASN A 218 209.199 82.856 196.804 1.0015.00 ATOM 1309 O ASN A 218 210.327 83.158 197.160 1.00 16.81 ATOM 1310N SER A 219 208.738 81.608 196.607 1.00 15.00 ATOM 1311 CA SER A 219209.612 80.496 196.960 1.00 15.00 ATOM 1312 CB SER A 219 208.946 79.616198.021 1.00 15.00 ATOM 1313 OG SER A 219 207.780 78.995 197.509 1.0015.00 ATOM 1314 C SER A 219 209.953 79.658 195.732 1.00 15.00 ATOM 1315O SER A 219 209.421 79.706 194.646 1.00 13.95 ATOM 1316 N LEU A 220211.012 78.785 195.962 1.00 15.00 ATOM 1317 CA LEU A 220 211.564 77.825195.013 1.00 15.00 ATOM 1318 CB LEU A 220 212.993 78.219 194.633 1.0015.00 ATOM 1319 CG LEU A 220 213.161 79.569 193.932 1.00 15.00 ATOM 1320CD1 LEU A 220 214.636 79.928 193.843 1.00 15.00 ATOM 1321 CD2 LEU A 220212.537 79.516 192.547 1.00 15.00 ATOM 1322 C LEU A 220 211.553 76.415195.594 1.00 15.00 ATOM 1323 O LEU A 220 211.679 76.279 196.816 1.0014.38 ATOM 1324 N THR A 221 211.526 75.374 194.729 1.00 15.00 ATOM 1325CA THR A 221 211.397 74.011 195.231 1.00 15.00 ATOM 1326 CB THR A 221210.079 73.365 194.763 1.00 15.00 ATOM 1327 OG1 THR A 221 208.970 74.121195.264 1.00 15.00 ATOM 1328 CG2 THR A 221 209.981 71.933 195.268 1.0015.00 ATOM 1329 C THR A 221 212.562 73.144 194.764 1.00 15.00 ATOM 1330O THR A 221 212.736 72.026 195.385 1.00 15.81 TER 1330 THR A 221 ATOM1331 N TPO A 222 213.399 73.479 193.800 1.00 15.00 ATOM 1332 CA TPO A222 214.459 72.638 193.257 1.00 15.00 ATOM 1333 CB TPO A 222 215.05673.250 191.976 1.00 15.00 ATOM 1334 OG1 TPO A 222 214.002 73.557 191.0551.00 15.00 ATOM 1335 CG2 TPO A 222 216.025 72.275 191.324 1.00 15.00ATOM 1336 P TPO A 222 213.808 74.828 190.542 1.00 15.80 P ATOM 1337 O1PTPO A 222 214.914 75.324 189.669 1.00 15.57 O ATOM 1338 O2P TPO A 222213.482 75.613 191.784 1.00 15.91 O ATOM 1339 O3P TPO A 222 212.59174.609 189.572 1.00 15.73 O ATOM 1340 C TPO A 222 215.578 72.444 194.2761.00 15.00 ATOM 1341 O TPO A 222 216.123 73.375 194.857 1.00 12.56 ATOM1342 N PRO A 223 215.917 71.098 194.555 1.00 15.00 ATOM 1343 CD PRO A223 215.209 69.889 194.261 1.00 15.00 ATOM 1344 CA PRO A 223 217.12170.849 195.355 1.00 15.00 ATOM 1345 CB PRO A 223 216.915 69.424 195.8681.00 15.00 ATOM 1346 CG PRO A 223 216.042 68.783 194.845 1.00 15.00 ATOM1347 C PRO A 223 218.396 70.958 194.525 1.00 15.00 ATOM 1348 O PRO A 223218.639 70.105 193.676 1.00 12.12 ATOM 1349 N CYS A 224 219.010 72.137194.641 1.00 15.00 ATOM 1350 CA CYS A 224 220.180 72.438 193.826 1.0015.00 ATOM 1351 CB CYS A 224 219.754 73.079 192.503 1.00 15.00 ATOM 1352SG CYS A 224 219.284 71.899 191.217 1.00 15.00 ATOM 1353 C CYS A 224221.138 73.367 194.565 1.00 15.00 ATOM 1354 O CYS A 224 220.831 73.947195.595 1.00 9.31 ATOM 1355 N TYR A 225 222.314 73.544 193.951 1.0015.00 ATOM 1356 CA TYR A 225 223.376 74.375 194.506 1.00 15.00 ATOM 1357CB TYR A 225 223.004 74.838 195.916 1.00 15.00 ATOM 1358 CG TYR A 225222.663 73.709 196.863 1.00 15.00 ATOM 1359 CD1 TYR A 225 223.543 72.652197.060 1.00 15.00 ATOM 1360 CE1 TYR A 225 223.236 71.620 197.924 1.0015.00 ATOM 1361 CD2 TYR A 225 221.461 73.699 197.556 1.00 15.00 ATOM1362 CE2 TYR A 225 221.146 72.667 198.422 1.00 15.00 ATOM 1363 CZ TYR A225 222.036 71.631 198.602 1.00 15.00 ATOM 1364 OH TYR A 225 221.72570.605 199.464 1.00 15.00 ATOM 1365 C TYR A 225 224.699 73.618 194.5431.00 15.00 ATOM 1366 O TYR A 225 224.872 72.695 193.743 1.00 11.69 ATOM1367 N THR A 226 225.564 74.018 195.383 1.00 15.00 ATOM 1368 CA THR A226 226.833 73.340 195.622 1.00 15.00 ATOM 1369 CB THR A 226 228.02674.279 195.362 1.00 15.00 ATOM 1370 OG1 THR A 226 229.137 73.880 196.1751.00 15.00 ATOM 1371 CG2 THR A 226 227.653 75.716 195.691 1.00 15.00ATOM 1372 C THR A 226 226.916 72.827 197.056 1.00 15.00 ATOM 1373 O THRA 226 226.576 73.621 197.967 1.00 9.55 ATOM 1374 N PRO A 227 227.14071.546 197.345 1.00 15.00 ATOM 1375 CD PRO A 227 228.192 71.043 196.5151.00 15.00 ATOM 1376 CA PRO A 227 227.065 70.837 198.626 1.00 15.00 ATOM1377 CB PRO A 227 227.911 69.587 198.389 1.00 15.00 ATOM 1378 CG PRO A227 228.885 69.992 197.336 1.00 15.00 ATOM 1379 C PRO A 227 227.62171.670 199.777 1.00 15.00 ATOM 1380 O PRO A 227 226.859 71.868 200.7941.00 10.86 ATOM 1381 N TYR A 228 228.838 72.168 199.777 1.00 15.00 ATOM1382 CA TYR A 228 229.509 72.872 200.863 1.00 15.00 ATOM 1383 CB TYR A228 230.999 73.029 200.552 1.00 15.00 ATOM 1384 CG TYR A 228 231.74871.718 200.461 1.00 15.00 ATOM 1385 CD1 TYR A 228 232.077 71.168 199.2281.00 15.00 ATOM 1386 CE1 TYR A 228 232.762 69.972 199.141 1.00 15.00ATOM 1387 CD2 TYR A 228 232.124 71.030 201.607 1.00 15.00 ATOM 1388 CE2TYR A 228 232.808 69.831 201.527 1.00 15.00 ATOM 1389 CZ TYR A 228233.124 69.307 200.293 1.00 15.00 ATOM 1390 OH TYR A 228 233.805 68.114200.210 1.00 15.00 ATOM 1391 C TYR A 228 228.883 74.243 201.095 1.0015.00 ATOM 1392 O TYR A 228 229.096 74.833 202.195 1.00 10.82 ATOM 1393N TYR A 229 228.075 74.767 200.161 1.00 15.00 ATOM 1394 CA TYR A 229227.513 76.111 200.221 1.00 15.00 ATOM 1395 CB TYR A 229 227.822 76.871198.929 1.00 15.00 ATOM 1396 CG TYR A 229 229.281 77.238 198.768 1.0015.00 ATOM 1397 CD1 TYR A 229 229.951 76.991 197.577 1.00 15.00 ATOM1398 CE1 TYR A 229 231.283 77.325 197.425 1.00 15.00 ATOM 1399 CD2 TYR A229 229.987 77.829 199.807 1.00 15.00 ATOM 1400 CE2 TYR A 229 231.32078.165 199.661 1.00 15.00 ATOM 1401 CZ TYR A 229 231.962 77.911 198.4701.00 15.00 ATOM 1402 OH TYR A 229 233.289 78.245 198.324 1.00 15.00 ATOM1403 C TYR A 229 226.005 76.064 200.443 1.00 15.00 ATOM 1404 O TYR A 229225.390 77.180 200.548 1.00 10.79 ATOM 1405 N VAL A 230 225.363 74.929200.540 1.00 15.00 ATOM 1406 CA VAL A 230 223.918 74.808 200.689 1.0015.00 ATOM 1407 CB VAL A 230 223.468 73.336 200.596 1.00 15.00 ATOM 1408CG1 VAL A 230 224.223 72.492 201.612 1.00 15.00 ATOM 1409 CG2 VAL A 230221.967 73.230 200.819 1.00 15.00 ATOM 1410 C VAL A 230 223.457 75.378202.027 1.00 15.00 ATOM 1411 O VAL A 230 224.048 75.172 203.074 1.009.09 ATOM 1412 CB ALA A 231 220.726 77.807 202.673 1.00 15.00 ATOM 1413C ALA A 231 220.871 75.607 203.851 1.00 15.00 ATOM 1414 O ALA A 231220.417 74.655 203.184 1.00 9.49 ATOM 1415 N ALA A 231 222.349 76.103201.918 1.00 15.00 ATOM 1416 CA ALA A 231 221.655 76.680 203.104 1.0015.00 ATOM 1417 N PRO A 232 220.755 75.645 205.185 1.00 15.00 ATOM 1418CD PRO A 232 220.885 76.873 205.909 1.00 15.00 ATOM 1419 CA PRO A 232220.064 74.616 205.968 1.00 15.00 ATOM 1420 CB PRO A 232 220.102 75.171207.392 1.00 15.00 ATOM 1421 CG PRO A 232 220.173 76.648 207.213 1.0015.00 ATOM 1422 C PRO A 232 218.629 74.402 205.498 1.00 15.00 ATOM 1423O PRO A 232 218.155 73.251 205.520 1.00 10.53 ATOM 1424 N GLU A 233217.931 75.458 205.055 1.00 15.00 ATOM 1425 CA GLU A 233 216.540 75.343204.633 1.00 15.00 ATOM 1426 CB GLU A 233 215.917 76.732 204.474 1.0015.00 ATOM 1427 CG GLU A 233 214.503 76.844 205.020 1.00 15.00 ATOM 1428CD GLU A 233 213.601 77.678 204.133 1.00 15.00 ATOM 1429 OE1 GLU A 233212.376 77.703 204.384 1.00 15.00 ATOM 1430 OE2 GLU A 233 214.114 78.309203.185 1.00 15.00 ATOM 1431 C GLU A 233 216.428 74.573 203.321 1.0015.00 ATOM 1432 O GLU A 233 215.392 73.897 203.095 1.00 10.95 ATOM 1433N VAL A 234 217.465 74.585 202.462 1.00 15.00 ATOM 1434 CA VAL A 234217.484 73.810 201.227 1.00 15.00 ATOM 1435 CB VAL A 234 218.603 74.293200.282 1.00 15.00 ATOM 1436 CG1 VAL A 234 218.713 73.363 199.084 1.0015.00 ATOM 1437 CG2 VAL A 234 218.331 75.720 199.832 1.00 15.00 ATOM1438 C VAL A 234 217.690 72.327 201.515 1.00 15.00 ATOM 1439 O VAL A 234217.255 71.447 200.787 1.00 10.86 ATOM 1440 N LEU A 235 218.265 72.046202.723 1.00 15.00 ATOM 1441 CA LEU A 235 218.512 70.679 203.169 1.0015.00 ATOM 1442 CB LEU A 235 219.760 70.629 204.053 1.00 15.00 ATOM 1443CG LEU A 235 221.077 71.046 203.392 1.00 15.00 ATOM 1444 CD1 LEU A 235222.165 71.181 204.445 1.00 15.00 ATOM 1445 CD2 LEU A 235 221.473 70.027202.337 1.00 15.00 ATOM 1446 C LEU A 235 217.313 70.129 203.934 1.0015.00 ATOM 1447 O LEU A 235 217.361 69.014 204.500 1.00 10.16 ATOM 1448N GLY A 236 216.252 70.887 203.946 1.00 15.00 ATOM 1449 CA GLY A 236214.958 70.501 204.475 1.00 15.00 ATOM 1450 C GLY A 236 213.924 70.292203.386 1.00 15.00 ATOM 1451 O GLY A 236 214.223 70.745 202.251 1.0015.43 ATOM 1452 N PRO A 237 212.800 69.571 203.652 1.00 15.00 ATOM 1453CD PRO A 237 212.368 68.973 204.879 1.00 15.00 ATOM 1454 CA PRO A 237211.813 69.401 202.581 1.00 15.00 ATOM 1455 CB PRO A 237 210.885 68.315203.124 1.00 15.00 ATOM 1456 CG PRO A 237 210.988 68.445 204.605 1.0015.00 ATOM 1457 C PRO A 237 211.047 70.688 202.295 1.00 15.00 ATOM 1458O PRO A 237 211.186 71.681 202.987 1.00 19.34 ATOM 1459 N GLU A 238210.242 70.629 201.243 1.00 15.00 ATOM 1460 CA GLU A 238 209.352 71.651200.704 1.00 15.00 ATOM 1461 CB GLU A 238 208.401 72.153 201.793 1.0015.00 ATOM 1462 CG GLU A 238 207.415 71.107 202.287 1.00 15.00 ATOM 1463CD GLU A 238 206.523 71.625 203.397 1.00 15.00 ATOM 1464 OE1 GLU A 238205.595 70.894 203.807 1.00 15.00 ATOM 1465 OE2 GLU A 238 206.747 72.764203.860 1.00 15.00 ATOM 1466 C GLU A 238 210.146 72.820 200.131 1.0015.00 ATOM 1467 O GLU A 238 211.191 72.629 199.449 1.00 25.64 ATOM 1468N LYS A 239 209.723 74.116 200.341 1.00 15.00 ATOM 1469 CA LYS A 239210.244 75.246 199.582 1.00 15.00 ATOM 1470 CB LYS A 239 209.094 76.047198.966 1.00 15.00 ATOM 1471 CG LYS A 239 208.369 75.328 197.840 1.0015.00 ATOM 1472 CD LYS A 239 207.259 76.189 197.261 1.00 15.00 ATOM 1473CE LYS A 239 206.534 75.469 196.134 1.00 15.00 ATOM 1474 NZ LYS A 239205.442 76.301 195.556 1.00 15.00 ATOM 1475 C LYS A 239 211.090 76.155200.466 1.00 15.00 ATOM 1476 O LYS A 239 211.196 76.171 201.667 1.0016.81 ATOM 1477 N TYR A 240 211.840 77.115 199.704 1.00 15.00 ATOM 1478CA TYR A 240 212.736 78.087 200.316 1.00 15.00 ATOM 1479 CB TYR A 240214.163 77.536 200.363 1.00 15.00 ATOM 1480 CG TYR A 240 214.299 76.138199.801 1.00 15.00 ATOM 1481 CD1 TYR A 240 214.813 75.929 198.526 1.0015.00 ATOM 1482 CE1 TYR A 240 214.940 74.655 198.009 1.00 15.00 ATOM1483 CD2 TYR A 240 213.913 75.030 200.542 1.00 15.00 ATOM 1484 CE2 TYR A240 214.038 73.752 200.030 1.00 15.00 ATOM 1485 CZ TYR A 240 214.55073.570 198.765 1.00 15.00 ATOM 1486 OH TYR A 240 214.674 72.298 198.2521.00 15.00 ATOM 1487 C TYR A 240 212.717 79.406 199.552 1.00 15.00 ATOM1488 O TYR A 240 212.436 79.469 198.354 1.00 13.90 ATOM 1489 N ASP A 241213.113 80.515 200.250 1.00 15.00 ATOM 1490 CA ASP A 241 213.048 81.860199.690 1.00 15.00 ATOM 1491 CB ASP A 241 212.482 82.836 200.725 1.0015.00 ATOM 1492 CG ASP A 241 211.007 82.608 200.994 1.00 15.00 ATOM 1493OD1 ASP A 241 210.289 82.190 200.062 1.00 15.00 ATOM 1494 OD2 ASP A 241210.567 82.847 202.138 1.00 15.00 ATOM 1495 C ASP A 241 214.426 82.330199.237 1.00 15.00 ATOM 1496 O ASP A 241 215.421 81.629 199.180 1.0012.46 ATOM 1497 N LYS A 242 214.597 83.587 198.906 1.00 106.74 N ATOM1498 CA LYS A 242 215.786 84.284 198.467 1.00 86.50 C ATOM 1499 C LYS A242 216.900 84.299 199.494 1.00 99.35 C ATOM 1500 O LYS A 242 217.94884.893 199.254 1.00 106.37 O ATOM 1501 CB LYS A 242 215.446 85.726198.107 1.00 97.76 C ATOM 1502 CG LYS A 242 214.108 86.236 198.608 1.0071.36 C ATOM 1503 CD LYS A 242 213.651 87.417 197.771 1.00 103.00 C ATOM1504 CE LYS A 242 212.555 88.212 198.477 1.00 138.19 C ATOM 1505 NZ LYSA 242 212.180 89.484 197.778 1.00 123.67 N ATOM 1506 N SER A 243 216.68183.659 200.637 1.00 97.75 N ATOM 1507 CA SER A 243 217.683 83.627201.704 1.00 84.81 C ATOM 1508 C SER A 243 218.986 82.926 201.333 1.0082.48 C ATOM 1509 O SER A 243 220.025 83.174 201.948 1.00 74.56 O ATOM1510 CB SER A 243 217.097 82.973 202.940 1.00 77.67 C ATOM 1511 OG SER A243 215.950 83.691 203.344 1.00 121.59 O ATOM 1512 N CYS A 244 218.94482.044 200.343 1.00 67.22 N ATOM 1513 CA CYS A 244 220.098 81.217200.052 1.00 78.41 C ATOM 1514 C CYS A 244 221.259 82.130 199.674 1.0064.58 C ATOM 1515 O CYS A 244 222.377 81.930 200.139 1.00 67.24 O ATOM1516 CB CYS A 244 219.790 80.254 198.911 1.00 64.99 C ATOM 1517 SG CYS A244 219.163 81.050 197.435 1.00 91.08 S ATOM 1518 N ASP A 245 220.98883.129 198.834 1.00 49.99 N ATOM 1519 CA ASP A 245 222.016 84.076198.416 1.00 45.00 C ATOM 1520 C ASP A 245 222.720 84.635 199.651 1.0060.39 C ATOM 1521 O ASP A 245 223.952 84.702 199.688 1.00 67.65 O ATOM1522 CB ASP A 245 221.410 85.236 197.620 1.00 69.55 C ATOM 1523 CG ASP A245 221.092 84.874 196.180 1.00 60.27 C ATOM 1524 OD1 ASP A 245 221.53583.802 195.715 1.00 73.75 O ATOM 1525 OD2 ASP A 245 220.410 85.679195.506 1.00 70.27 O ATOM 1526 N MET A 246 221.937 85.033 200.659 1.0043.54 N ATOM 1527 CA MET A 246 222.494 85.577 201.898 1.00 64.86 C ATOM1528 C MET A 246 223.266 84.526 202.684 1.00 67.48 C ATOM 1529 O MET A246 224.198 84.855 203.425 1.00 67.31 O ATOM 1530 CB MET A 246 221.40186.146 202.790 1.00 52.62 C ATOM 1531 CG MET A 246 220.635 87.263202.163 1.00 60.79 C ATOM 1532 SD MET A 246 221.708 88.445 201.385 1.0066.30 S ATOM 1533 CE MET A 246 222.403 89.309 202.804 1.00 55.43 C ATOM1534 N TRP A 247 222.868 83.265 202.536 1.00 54.14 N ATOM 1535 CA TRP A247 223.551 82.177 203.215 1.00 62.36 C ATOM 1536 C TRP A 247 224.93482.015 202.592 1.00 62.90 C ATOM 1537 O TRP A 247 225.951 82.071 203.2871.00 62.42 O ATOM 1538 CB TRP A 247 222.748 80.888 203.074 1.00 69.04 CATOM 1539 CG TRP A 247 223.470 79.670 203.559 1.00 86.20 C ATOM 1540 CD1TRP A 247 224.327 78.896 202.844 1.00 72.11 C ATOM 1541 CD2 TRP A 247223.430 79.110 204.885 1.00 75.65 C ATOM 1542 NE1 TRP A 247 224.82777.885 203.636 1.00 81.90 N ATOM 1543 CE2 TRP A 247 224.293 77.999204.893 1.00 68.57 C ATOM 1544 CE3 TRP A 247 222.749 79.446 206.064 1.0091.13 C ATOM 1545 CZ2 TRP A 247 224.500 77.221 206.030 1.00 68.02 C ATOM1546 CZ3 TRP A 247 222.952 78.669 207.193 1.00 82.48 C ATOM 1547 CH2 TRPA 247 223.821 77.572 207.166 1.00 74.62 C ATOM 1548 N SER A 248 224.95981.829 201.274 1.00 77.02 N ATOM 1549 CA SER A 248 226.203 81.676200.531 1.00 73.53 C ATOM 1550 C SER A 248 227.156 82.786 200.892 1.0080.75 C ATOM 1551 O SER A 248 228.369 82.583 200.943 1.00 84.06 O ATOM1552 CB SER A 248 225.930 81.731 199.043 1.00 67.92 C ATOM 1553 OG SER A248 225.112 80.642 198.668 1.00 118.25 O ATOM 1554 N LEU A 249 226.59383.967 201.130 1.00 76.04 N ATOM 1555 CA LEU A 249 227.378 85.132201.506 1.00 82.09 C ATOM 1556 C LEU A 249 228.081 84.869 202.845 1.0084.27 C ATOM 1557 O LEU A 249 229.298 85.035 202.960 1.00 83.90 O ATOM1558 CB LEU A 249 226.473 86.363 201.610 1.00 85.62 C ATOM 1559 CG LEU A249 227.049 87.629 200.964 1.00 77.42 C ATOM 1560 CD1 LEU A 249 227.25087.346 199.493 1.00 68.42 C ATOM 1561 CD2 LEU A 249 226.119 88.832201.153 1.00 95.64 C ATOM 1562 N GLY A 250 227.318 84.449 203.851 1.0064.22 N ATOM 1563 CA GLY A 250 227.915 84.165 205.147 1.00 62.72 C ATOM1564 C GLY A 250 229.103 83.231 204.998 1.00 63.71 C ATOM 1565 O GLY A250 230.220 83.544 205.418 1.00 57.98 O ATOM 1566 N VAL A 251 228.85582.076 204.387 1.00 57.35 N ATOM 1567 CA VAL A 251 229.902 81.091204.152 1.00 72.77 C ATOM 1568 C VAL A 251 231.130 81.779 203.587 1.0065.76 C ATOM 1569 O VAL A 251 232.239 81.624 204.093 1.00 72.08 O ATOM1570 CB VAL A 251 229.474 80.052 203.129 1.00 72.02 C ATOM 1571 CG1 VALA 251 230.627 79.119 202.852 1.00 48.81 C ATOM 1572 CG2 VAL A 251228.255 79.312 203.617 1.00 45.51 C ATOM 1573 N ILE A 252 230.927 82.544202.525 1.00 65.58 N ATOM 1574 CA ILE A 252 232.036 83.248 201.899 1.0068.61 C ATOM 1575 C ILE A 252 232.722 84.197 202.876 1.00 77.12 C ATOM1576 O ILE A 252 233.932 84.098 203.088 1.00 79.18 O ATOM 1577 CB ILE A252 231.564 84.008 200.634 1.00 65.58 C ATOM 1578 CG1 ILE A 252 231.15182.989 199.570 1.00 42.53 C ATOM 1579 CG2 ILE A 252 232.679 84.891200.102 1.00 53.46 C ATOM 1580 CD1 ILE A 252 230.557 83.587 198.355 1.0059.04 C ATOM 1581 N MET A 253 231.956 85.091 203.498 1.00 72.95 N ATOM1582 CA MET A 253 232.553 86.028 204.444 1.00 81.03 C ATOM 1583 C MET A253 233.367 85.286 205.497 1.00 75.68 C ATOM 1584 O MET A 253 234.46085.724 205.883 1.00 79.62 O ATOM 1585 CB MET A 253 231.486 86.866205.149 1.00 64.17 C ATOM 1586 CG MET A 253 232.083 88.081 205.819 1.0054.30 C ATOM 1587 SD MET A 253 230.954 88.969 206.885 1.00 85.00 S ATOM1588 CE MET A 253 229.555 89.250 205.802 1.00 83.83 C ATOM 1589 N TYR A254 232.829 84.161 205.960 1.00 81.14 N ATOM 1590 CA TYR A 254 233.51383.370 206.969 1.00 68.03 C ATOM 1591 C TYR A 254 234.879 83.008 206.4191.00 74.50 C ATOM 1592 O TYR A 254 235.901 83.519 206.879 1.00 78.65 OATOM 1593 CB TYR A 254 232.711 82.107 207.279 1.00 66.09 C ATOM 1594 CGTYR A 254 233.253 81.291 208.427 1.00 68.80 C ATOM 1595 CD1 TYR A 254234.285 80.388 208.230 1.00 93.85 C ATOM 1596 CD2 TYR A 254 232.73981.434 209.713 1.00 66.37 C ATOM 1597 CE1 TYR A 254 234.797 79.640209.281 1.00 93.85 C ATOM 1598 CE2 TYR A 254 233.238 80.696 210.775 1.0060.25 C ATOM 1599 CZ TYR A 254 234.270 79.797 210.553 1.00 78.16 C ATOM1600 OH TYR A 254 234.780 79.043 211.592 1.00 83.85 O ATOM 1601 N ILE A255 234.876 82.148 205.405 1.00 71.34 N ATOM 1602 CA ILE A 255 236.09981.690 204.774 1.00 61.37 C ATOM 1603 C ILE A 255 237.128 82.782 204.5581.00 76.55 C ATOM 1604 O ILE A 255 238.298 82.590 204.870 1.00 92.35 OATOM 1605 CB ILE A 255 235.810 81.046 203.443 1.00 62.35 C ATOM 1606 CG1ILE A 255 234.928 79.819 203.653 1.00 62.67 C ATOM 1607 CG2 ILE A 255237.107 80.661 202.790 1.00 76.47 C ATOM 1608 CD1 ILE A 255 234.51479.131 202.387 1.00 57.99 C ATOM 1609 N LEU A 256 236.709 83.925 204.0241.00 73.36 N ATOM 1610 CA LEU A 256 237.647 85.022 203.809 1.00 76.61 CATOM 1611 C LEU A 256 238.466 85.295 205.065 1.00 80.02 C ATOM 1612 OLEU A 256 239.681 85.116 205.074 1.00 95.93 O ATOM 1613 CB LEU A 256236.921 86.316 203.430 1.00 82.53 C ATOM 1614 CG LEU A 256 236.50386.623 201.991 1.00 67.94 C ATOM 1615 CD1 LEU A 256 236.199 88.119201.870 1.00 83.50 C ATOM 1616 CD2 LEU A 256 237.615 86.248 201.038 1.0061.15 C ATOM 1617 N LEU A 257 237.780 85.718 206.124 1.00 64.99 N ATOM1618 CA LEU A 257 238.407 86.060 207.399 1.00 83.07 C ATOM 1619 C LEU A257 239.336 85.046 208.090 1.00 92.07 C ATOM 1620 O LEU A 257 240.17585.450 208.897 1.00 79.63 O ATOM 1621 CB LEU A 257 237.331 86.467208.402 1.00 65.07 C ATOM 1622 CG LEU A 257 236.445 87.661 208.054 1.0072.20 C ATOM 1623 CD1 LEU A 257 235.475 87.888 209.185 1.00 109.20 CATOM 1624 CD2 LEU A 257 237.282 88.906 207.843 1.00 77.76 C ATOM 1625 NCYS A 258 239.211 83.751 207.805 1.00 84.06 N ATOM 1626 CA CYS A 258240.068 82.782 208.486 1.00 65.38 C ATOM 1627 C CYS A 258 240.811 81.822207.572 1.00 79.35 C ATOM 1628 O CYS A 258 241.865 81.305 207.936 1.0089.35 O ATOM 1629 CB CYS A 258 239.250 81.952 209.465 1.00 74.82 C ATOM1630 SG CYS A 258 238.333 80.661 208.627 1.00 88.33 S ATOM 1631 N GLY A259 240.259 81.552 206.397 1.00 77.77 N ATOM 1632 CA GLY A 259 240.92780.636 205.491 1.00 86.18 C ATOM 1633 C GLY A 259 240.211 79.311 205.3061.00 84.64 C ATOM 1634 O GLY A 259 240.562 78.538 204.415 1.00 91.47 OATOM 1635 N TYR A 260 239.219 79.030 206.145 1.00 80.25 N ATOM 1636 CATYR A 260 238.454 77.788 206.024 1.00 85.40 C ATOM 1637 C TYR A 260236.969 78.064 206.208 1.00 92.78 C ATOM 1638 O TYR A 260 236.587 79.064206.813 1.00 109.20 O ATOM 1639 CB TYR A 260 238.936 76.760 207.050 1.0090.73 C ATOM 1640 CG TYR A 260 239.030 77.305 208.451 1.00 78.95 C ATOM1641 CD1 TYR A 260 237.942 77.261 209.312 1.00 99.14 C ATOM 1642 CD2 TYRA 260 240.204 77.892 208.904 1.00 82.37 C ATOM 1643 CE1 TYR A 260238.023 77.785 210.585 1.00 88.16 C ATOM 1644 CE2 TYR A 260 240.29278.420 210.173 1.00 97.24 C ATOM 1645 CZ TYR A 260 239.201 78.364211.007 1.00 67.63 C ATOM 1646 OH TYR A 260 239.298 78.896 212.269 1.0084.01 O ATOM 1647 N PRO A 261 236.114 77.183 205.676 1.00 89.49 N ATOM1648 CA PRO A 261 234.656 77.296 205.753 1.00 103.35 C ATOM 1649 C PRO A261 234.107 77.095 207.161 1.00 96.36 C ATOM 1650 O PRO A 261 234.74376.459 207.995 1.00 100.89 O ATOM 1651 CB PRO A 261 234.185 76.209204.791 1.00 98.88 C ATOM 1652 CG PRO A 261 235.208 75.152 204.996 1.0095.80 C ATOM 1653 CD PRO A 261 236.493 75.962 204.950 1.00 91.52 C ATOM1654 N PRO A 262 232.918 77.653 207.444 1.00 94.45 N ATOM 1655 CA PRO A262 232.337 77.488 208.773 1.00 85.15 C ATOM 1656 C PRO A 262 232.01576.028 209.043 1.00 68.20 C ATOM 1657 O PRO A 262 232.519 75.448 209.9981.00 106.94 O ATOM 1658 CB PRO A 262 231.092 78.368 208.717 1.00 75.57 CATOM 1659 CG PRO A 262 230.721 78.337 207.278 1.00 64.63 C ATOM 1660 CDPRO A 262 232.053 78.504 206.609 1.00 99.03 C ATOM 1661 N PHE A 263231.200 75.419 208.191 1.00 73.57 N ATOM 1662 CA PHE A 263 230.83874.021 208.393 1.00 79.36 C ATOM 1663 C PHE A 263 231.864 73.093 207.7571.00 106.15 C ATOM 1664 O PHE A 263 231.972 73.009 206.530 1.00 105.28 OATOM 1665 CB PHE A 263 229.428 73.777 207.848 1.00 69.85 C ATOM 1666 CGPHE A 263 228.420 74.759 208.374 1.00 81.28 C ATOM 1667 CD1 PHE A 263228.128 75.917 207.677 1.00 113.87 C ATOM 1668 CD2 PHE A 263 227.84574.573 209.613 1.00 117.67 C ATOM 1669 CE1 PHE A 263 227.285 76.867208.205 1.00 85.69 C ATOM 1670 CE2 PHE A 263 227.004 75.520 210.145 1.00108.39 C ATOM 1671 CZ PHE A 263 226.729 76.670 209.437 1.00 117.73 CATOM 1672 N TYR A 264 232.620 72.408 208.622 1.00 134.25 N ATOM 1673 CATYR A 264 233.681 71.497 208.197 1.00 147.77 C ATOM 1674 C TYR A 264233.192 70.094 207.848 1.00 140.09 C ATOM 1675 O TYR A 264 232.00669.787 207.979 1.00 138.93 O ATOM 1676 CB TYR A 264 234.775 71.405209.284 1.00 153.86 C ATOM 1677 CG TYR A 264 236.191 71.629 208.760 1.00157.09 C ATOM 1678 CD1 TYR A 264 237.265 70.886 209.250 1.00 193.79 CATOM 1679 CD2 TYR A 264 236.448 72.574 207.768 1.00 140.55 C ATOM 1680CE1 TYR A 264 238.553 71.074 208.764 1.00 220.80 C ATOM 1681 CE2 TYR A264 237.734 72.771 207.279 1.00 201.56 C ATOM 1682 CZ TYR A 264 238.78272.015 207.781 1.00 222.82 C ATOM 1683 OH TYR A 264 240.069 72.185207.307 1.00 220.52 O ATOM 1684 N SER A 265 234.118 69.246 207.409 1.00141.69 N ATOM 1685 CA SER A 265 233.785 67.877 207.044 1.00 135.35 CATOM 1686 C SER A 265 234.560 66.896 207.915 1.00 131.47 C ATOM 1687 OSER A 265 235.792 66.945 207.976 1.00 126.66 O ATOM 1688 CB SER A 265234.113 67.628 205.566 1.00 138.65 C ATOM 1689 OG SER A 265 233.59166.386 205.130 1.00 107.77 O TER 1689 SER A 265 ATOM 1690 N GLY A 274229.307 63.721 209.431 1.00 106.41 N ATOM 1691 CA GLY A 274 228.03064.307 209.816 1.00 119.68 C ATOM 1692 C GLY A 274 227.987 65.812209.615 1.00 113.16 C ATOM 1693 O GLY A 274 227.553 66.551 210.496 1.0095.55 O ATOM 1694 N MET A 275 228.446 66.261 208.449 1.00 110.29 N ATOM1695 CA MET A 275 228.463 67.676 208.112 1.00 95.81 C ATOM 1696 C MET A275 227.067 68.162 207.746 1.00 94.19 C ATOM 1697 O MET A 275 226.63369.206 208.220 1.00 75.71 O ATOM 1698 CB MET A 275 229.412 67.938206.943 1.00 90.51 C ATOM 1699 CG MET A 275 229.404 69.380 206.472 1.0083.11 C ATOM 1700 SD MET A 275 230.324 69.611 204.942 1.00 99.84 S ATOM1701 CE MET A 275 229.028 69.352 203.750 1.00 79.14 C ATOM 1702 N LYS A276 226.362 67.411 206.904 1.00 80.06 N ATOM 1703 CA LYS A 276 225.01867.816 206.511 1.00 81.42 C ATOM 1704 C LYS A 276 224.233 68.145 207.7701.00 67.18 C ATOM 1705 O LYS A 276 223.297 68.931 207.738 1.00 87.18 OATOM 1706 CB LYS A 276 224.327 66.705 205.731 1.00 70.05 C ATOM 1707 NTHR A 277 224.641 67.552 208.886 1.00 90.20 N ATOM 1708 CA THR A 277223.982 67.776 210.167 1.00 86.79 C ATOM 1709 C THR A 277 224.402 69.103210.790 1.00 88.87 C ATOM 1710 O THR A 277 223.570 69.987 211.004 1.0090.25 O ATOM 1711 CB THR A 277 224.316 66.657 211.170 1.00 94.65 C ATOM1712 OG1 THR A 277 223.953 65.387 210.614 1.00 124.83 O ATOM 1713 CG2THR A 277 223.559 66.871 212.464 1.00 101.58 C ATOM 1714 N ARG A 278225.696 69.224 211.083 1.00 83.15 N ATOM 1715 CA ARG A 278 226.27370.425 211.683 1.00 85.59 C ATOM 1716 C ARG A 278 225.653 71.666 211.0391.00 80.18 C ATOM 1717 O ARG A 278 225.537 72.716 211.673 1.00 83.67 OATOM 1718 CB ARG A 278 227.792 70.413 211.479 1.00 76.56 C ATOM 1719 CGARG A 278 228.346 68.998 211.313 1.00 94.91 C ATOM 1720 CD ARG A 278229.398 68.600 212.352 1.00 109.01 C ATOM 1721 NE ARG A 278 230.69969.199 212.072 1.00 121.75 N ATOM 1722 CZ ARG A 278 231.384 69.003210.944 1.00 131.02 C ATOM 1723 NH1 ARG A 278 230.892 68.216 209.9881.00 101.83 N ATOM 1724 NH2 ARG A 278 232.556 69.613 210.763 1.00 135.84N ATOM 1725 N ILE A 279 225.250 71.516 209.778 1.00 83.10 N ATOM 1726 CAILE A 279 224.615 72.579 209.001 1.00 82.30 C ATOM 1727 C ILE A 279223.167 72.765 209.421 1.00 76.46 C ATOM 1728 O ILE A 279 222.778 73.847209.847 1.00 90.20 O ATOM 1729 CB ILE A 279 224.609 72.257 207.498 1.0084.92 C ATOM 1730 CG1 ILE A 279 226.006 72.436 206.913 1.00 77.12 C ATOM1731 CG2 ILE A 279 223.611 73.144 206.789 1.00 57.01 C ATOM 1732 CD1 ILEA 279 226.093 72.021 205.455 1.00 73.72 C ATOM 1733 N ARG A 280 222.37171.709 209.270 1.00 68.55 N ATOM 1734 CA ARG A 280 220.965 71.757209.640 1.00 66.51 C ATOM 1735 C ARG A 280 220.886 72.218 211.090 1.0062.26 C ATOM 1736 O ARG A 280 220.039 73.038 211.456 1.00 69.34 O ATOM1737 CB ARG A 280 220.326 70.383 209.475 1.00 49.45 C ATOM 1738 N MET A281 221.787 71.693 211.912 1.00 62.31 N ATOM 1739 CA MET A 281 221.84672.058 213.323 1.00 71.30 C ATOM 1740 C MET A 281 222.348 73.489 213.4381.00 78.13 C ATOM 1741 O MET A 281 222.151 74.152 214.450 1.00 79.48 OATOM 1742 CB MET A 281 222.794 71.122 214.075 1.00 73.13 C ATOM 1743 CGMET A 281 222.253 69.721 214.315 1.00 98.38 C ATOM 1744 SD MET A 281220.850 69.703 215.434 1.00 82.33 S ATOM 1745 CE MET A 281 221.65970.065 216.963 1.00 94.31 C ATOM 1746 N GLY A 282 223.000 73.953 212.3791.00 80.51 N ATOM 1747 CA GLY A 282 223.536 75.298 212.354 1.00 70.03 CATOM 1748 C GLY A 282 224.577 75.481 213.435 1.00 89.35 C ATOM 1749 OGLY A 282 224.566 76.482 214.146 1.00 77.94 O ATOM 1750 N GLN A 283225.479 74.514 213.576 1.00 81.75 N ATOM 1751 CA GLN A 283 226.50974.624 214.602 1.00 104.61 C ATOM 1752 C GLN A 283 227.939 74.764214.094 1.00 94.71 C ATOM 1753 O GLN A 283 228.503 73.843 213.505 1.0088.07 O ATOM 1754 CB GLN A 283 226.421 73.450 215.583 1.00 113.16 C ATOM1755 CG GLN A 283 226.575 72.075 214.982 1.00 114.78 C ATOM 1756 CD GLNA 283 226.574 71.002 216.057 1.00 133.39 C ATOM 1757 OE1 GLN A 283227.356 71.068 217.005 1.00 133.52 O ATOM 1758 NE2 GLN A 283 225.69170.012 215.922 1.00 112.55 N ATOM 1759 N TYR A 284 228.516 75.935214.347 1.00 90.04 N ATOM 1760 CA TYR A 284 229.875 76.242 213.934 1.0064.09 C ATOM 1761 C TYR A 284 230.474 77.137 215.008 1.00 65.66 C ATOM1762 O TYR A 284 229.806 77.468 215.981 1.00 88.03 O ATOM 1763 CB TYR A284 229.864 77.002 212.620 1.00 82.88 C ATOM 1764 CG TYR A 284 229.04078.259 212.690 1.00 50.94 C ATOM 1765 CD1 TYR A 284 227.656 78.208212.619 1.00 72.36 C ATOM 1766 CD2 TYR A 284 229.645 79.496 212.856 1.0079.70 C ATOM 1767 CE1 TYR A 284 226.901 79.355 212.709 1.00 69.83 C ATOM1768 CE2 TYR A 284 228.896 80.646 212.951 1.00 57.96 C ATOM 1769 CZ TYRA 284 227.527 80.569 212.877 1.00 57.53 C ATOM 1770 OH TYR A 284 226.77881.716 212.978 1.00 94.22 O ATOM 1771 N GLU A 285 231.725 77.538 214.8211.00 80.73 N ATOM 1772 CA GLU A 285 232.399 78.388 215.785 1.00 81.22 CATOM 1773 C GLU A 285 233.397 79.305 215.101 1.00 90.67 C ATOM 1774 OGLU A 285 233.713 79.119 213.926 1.00 106.16 O ATOM 1775 CB GLU A 285233.113 77.527 216.807 1.00 87.46 C ATOM 1776 N PHE A 286 233.875 80.303215.839 1.00 86.81 N ATOM 1777 CA PHE A 286 234.880 81.236 215.334 1.0097.06 C ATOM 1778 C PHE A 286 236.110 80.887 216.167 1.00 100.16 C ATOM1779 O PHE A 286 236.527 81.662 217.028 1.00 110.05 O ATOM 1780 CB PHE A286 234.476 82.697 215.602 1.00 78.01 C ATOM 1781 CG PHE A 286 233.21483.127 214.904 1.00 98.13 C ATOM 1782 CD1 PHE A 286 232.172 83.698215.615 1.00 92.96 C ATOM 1783 CD2 PHE A 286 233.064 82.959 213.538 1.00102.41 C ATOM 1784 CE1 PHE A 286 231.003 84.092 214.980 1.00 96.16 CATOM 1785 CE2 PHE A 286 231.892 83.353 212.897 1.00 82.36 C ATOM 1786 CZPHE A 286 230.865 83.918 213.620 1.00 71.90 C ATOM 1787 N PRO A 287236.707 79.715 215.909 1.00 96.70 N ATOM 1788 CA PRO A 287 237.88379.170 216.586 1.00 103.18 C ATOM 1789 C PRO A 287 239.086 80.081216.730 1.00 112.32 C ATOM 1790 O PRO A 287 239.531 80.709 215.770 1.00114.13 O ATOM 1791 CB PRO A 287 238.210 77.940 215.754 1.00 105.25 CATOM 1792 CG PRO A 287 237.828 78.359 214.411 1.00 92.44 C ATOM 1793 CDPRO A 287 236.474 78.978 214.659 1.00 100.51 C ATOM 1794 N ASN A 288239.608 80.133 217.950 1.00 119.25 N ATOM 1795 CA ASN A 288 240.79680.913 218.239 1.00 131.90 C ATOM 1796 C ASN A 288 241.953 79.964217.949 1.00 138.51 C ATOM 1797 O ASN A 288 241.808 78.742 218.021 1.00143.95 O ATOM 1798 CB ASN A 288 240.826 81.347 219.708 1.00 133.89 CATOM 1799 CG ASN A 288 239.631 82.211 220.092 1.00 158.92 C ATOM 1800OD1 ASN A 288 239.383 83.259 219.485 1.00 172.68 O ATOM 1801 ND2 ASN A288 238.890 81.779 221.114 1.00 139.14 N ATOM 1802 N PRO A 289 243.11980.510 217.602 1.00 145.89 N ATOM 1803 CA PRO A 289 243.399 81.940217.477 1.00 147.78 C ATOM 1804 C PRO A 289 242.632 82.643 216.367 1.00140.12 C ATOM 1805 O PRO A 289 242.027 83.678 216.608 1.00 145.48 O ATOM1806 CB PRO A 289 244.904 81.967 217.231 1.00 160.27 C ATOM 1807 CG PROA 289 245.107 80.724 216.415 1.00 158.40 C ATOM 1808 CD PRO A 289244.286 79.712 217.185 1.00 149.68 C ATOM 1809 N GLU A 290 242.66582.064 215.166 1.00 135.89 N ATOM 1810 CA GLU A 290 242.019 82.602213.959 1.00 122.33 C ATOM 1811 C GLU A 290 241.100 83.810 214.141 1.00111.48 C ATOM 1812 O GLU A 290 241.314 84.858 213.531 1.00 94.90 O ATOM1813 CB GLU A 290 241.230 81.502 213.236 1.00 121.58 C ATOM 1814 CG GLUA 290 241.934 80.155 213.147 1.00 140.70 C ATOM 1815 CD GLU A 290241.650 79.287 214.360 1.00 183.86 C ATOM 1816 OE1 GLU A 290 241.89879.762 215.484 1.00 193.44 O ATOM 1817 OE2 GLU A 290 241.178 78.137214.193 1.00 182.20 O ATOM 1818 N TRP A 291 240.080 83.655 214.980 1.00102.18 N ATOM 1819 CA TRP A 291 239.105 84.715 215.221 1.00 106.95 CATOM 1820 C TRP A 291 239.335 85.573 216.457 1.00 115.40 C ATOM 1821 OTRP A 291 238.474 86.375 216.824 1.00 120.18 O ATOM 1822 CB TRP A 291237.712 84.106 215.295 1.00 110.40 C ATOM 1823 CG TRP A 291 237.36483.374 214.056 1.00 104.11 C ATOM 1824 CD1 TRP A 291 237.782 82.121213.692 1.00 84.92 C ATOM 1825 CD2 TRP A 291 236.556 83.857 212.982 1.00106.19 C ATOM 1826 NE1 TRP A 291 237.282 81.799 212.456 1.00 103.99 NATOM 1827 CE2 TRP A 291 236.529 82.848 211.996 1.00 102.71 C ATOM 1828CE3 TRP A 291 235.857 85.048 212.753 1.00 70.56 C ATOM 1829 CZ2 TRP A291 235.827 82.992 210.807 1.00 86.03 C ATOM 1830 CZ3 TRP A 291 235.16585.189 211.578 1.00 73.88 C ATOM 1831 CH2 TRP A 291 235.154 84.171210.616 1.00 114.55 C ATOM 1832 N SER A 292 240.491 85.404 217.091 1.00129.00 N ATOM 1833 CA SER A 292 240.851 86.158 218.287 1.00 124.04 CATOM 1834 C SER A 292 240.910 87.670 218.051 1.00 123.03 C ATOM 1835 OSER A 292 240.661 88.455 218.966 1.00 113.67 O ATOM 1836 CB SER A 292242.199 85.658 218.825 1.00 125.10 C ATOM 1837 OG SER A 292 243.19685.669 217.817 1.00 135.63 O ATOM 1838 N GLU A 293 241.237 88.081216.829 1.00 123.41 N ATOM 1839 CA GLU A 293 241.321 89.508 216.513 1.00127.66 C ATOM 1840 C GLU A 293 240.108 90.026 215.724 1.00 121.92 C ATOM1841 O GLU A 293 240.091 91.178 215.285 1.00 115.91 O ATOM 1842 CB GLU A293 242.600 89.792 215.737 1.00 138.28 C ATOM 1843 N VAL A 294 239.09489.181 215.555 1.00 111.91 N ATOM 1844 CA VAL A 294 237.898 89.568214.818 1.00 95.15 C ATOM 1845 C VAL A 294 236.811 90.136 215.721 1.00103.25 C ATOM 1846 O VAL A 294 236.506 89.573 216.771 1.00 98.28 O ATOM1847 CB VAL A 294 237.322 88.378 214.066 1.00 92.93 C ATOM 1848 CG1 VALA 294 236.182 88.840 213.168 1.00 91.77 C ATOM 1849 CG2 VAL A 294238.418 87.705 213.269 1.00 94.47 C ATOM 1850 N SER A 295 236.218 91.249215.292 1.00 84.68 N ATOM 1851 CA SER A 295 235.168 91.914 216.056 1.0092.73 C ATOM 1852 C SER A 295 233.919 91.072 216.244 1.00 88.74 C ATOM1853 O SER A 295 233.568 90.250 215.399 1.00 88.58 O ATOM 1854 CB SER A295 234.745 93.201 215.365 1.00 102.34 C ATOM 1855 OG SER A 295 233.46493.030 214.784 1.00 114.40 O ATOM 1856 N GLU A 296 233.244 91.301217.362 1.00 92.37 N ATOM 1857 CA GLU A 296 232.008 90.600 217.658 1.00104.61 C ATOM 1858 C GLU A 296 230.970 91.166 216.694 1.00 112.98 C ATOM1859 O GLU A 296 229.956 90.523 216.406 1.00 105.91 O ATOM 1860 CB GLU A296 231.593 90.857 219.108 1.00 99.76 C ATOM 1861 CG GLU A 296 230.31690.154 219.543 1.00 121.36 C ATOM 1862 CD GLU A 296 230.324 88.663219.236 1.00 136.85 C ATOM 1863 OE1 GLU A 296 231.304 87.970 219.6091.00 131.43 O ATOM 1864 OE2 GLU A 296 229.340 88.185 218.619 1.00 136.46O ATOM 1865 N GLU A 297 231.237 92.379 216.203 1.00 112.50 N ATOM 1866CA GLU A 297 230.357 93.045 215.247 1.00 99.85 C ATOM 1867 C GLU A 297230.420 92.241 213.960 1.00 92.47 C ATOM 1868 O GLU A 297 229.396 91.854213.400 1.00 93.48 O ATOM 1869 CB GLU A 297 230.838 94.472 214.998 1.0098.76 C ATOM 1870 CG GLU A 297 230.226 95.146 213.787 1.00 102.64 C ATOM1871 CD GLU A 297 230.489 96.642 213.773 1.00 129.34 C ATOM 1872 OE1 GLUA 297 229.777 97.379 214.497 1.00 134.48 O ATOM 1873 OE2 GLU A 297231.415 97.081 213.049 1.00 104.72 O ATOM 1874 N VAL A 298 231.63891.993 213.499 1.00 80.38 N ATOM 1875 CA VAL A 298 231.843 91.200212.305 1.00 87.49 C ATOM 1876 C VAL A 298 231.282 89.809 212.579 1.0080.88 C ATOM 1877 O VAL A 298 230.690 89.176 211.702 1.00 71.80 O ATOM1878 CB VAL A 298 233.333 91.057 211.993 1.00 64.02 C ATOM 1879 CG1 VALA 298 233.542 89.953 210.980 1.00 84.65 C ATOM 1880 CG2 VAL A 298233.873 92.364 211.466 1.00 112.61 C ATOM 1881 N LYS A 299 231.47989.331 213.801 1.00 79.83 N ATOM 1882 CA LYS A 299 230.990 88.015214.162 1.00 75.16 C ATOM 1883 C LYS A 299 229.478 87.983 214.138 1.0080.77 C ATOM 1884 O LYS A 299 228.872 87.052 213.618 1.00 62.14 O ATOM1885 CB LYS A 299 231.517 87.616 215.539 1.00 74.57 C ATOM 1886 CG LYS A299 232.979 87.229 215.509 1.00 75.11 C ATOM 1887 CD LYS A 299 233.43286.563 216.790 1.00 92.06 C ATOM 1888 CE LYS A 299 234.841 86.005216.623 1.00 125.81 C ATOM 1889 NZ LYS A 299 235.368 85.378 217.867 1.00126.47 N ATOM 1890 N MET A 300 228.872 89.021 214.691 1.00 78.00 N ATOM1891 CA MET A 300 227.428 89.116 214.735 1.00 80.36 C ATOM 1892 C MET A300 226.822 89.120 213.327 1.00 69.48 C ATOM 1893 O MET A 300 225.73288.587 213.118 1.00 75.70 O ATOM 1894 CB MET A 300 227.033 90.378215.502 1.00 87.97 C ATOM 1895 CG MET A 300 226.011 90.131 216.601 1.00118.90 C ATOM 1896 SD MET A 300 226.231 88.508 217.393 1.00 128.71 SATOM 1897 CE MET A 300 224.516 87.872 217.267 1.00 113.92 C ATOM 1898 NLEU A 301 227.538 89.707 212.369 1.00 73.89 N ATOM 1899 CA LEU A 301227.079 89.782 210.972 1.00 63.96 C ATOM 1900 C LEU A 301 227.048 88.406210.301 1.00 70.75 C ATOM 1901 O LEU A 301 226.145 88.086 209.532 1.0060.38 O ATOM 1902 CB LEU A 301 227.977 90.735 210.177 1.00 63.62 C ATOM1903 CG LEU A 301 227.617 91.019 208.720 1.00 52.63 C ATOM 1904 CD1 LEUA 301 226.130 91.284 208.596 1.00 65.57 C ATOM 1905 CD2 LEU A 301228.414 92.212 208.225 1.00 73.24 C ATOM 1906 N ILE A 302 228.046 87.588210.592 1.00 67.57 N ATOM 1907 CA ILE A 302 228.090 86.250 210.037 1.0061.70 C ATOM 1908 C ILE A 302 226.900 85.455 210.582 1.00 75.97 C ATOM1909 O ILE A 302 226.226 84.741 209.837 1.00 58.38 O ATOM 1910 CB ILE A302 229.422 85.565 210.402 1.00 70.53 C ATOM 1911 CG1 ILE A 302 230.56286.271 209.659 1.00 76.98 C ATOM 1912 CG2 ILE A 302 229.369 84.086210.071 1.00 65.01 C ATOM 1913 CD1 ILE A 302 231.930 85.670 209.869 1.0071.99 C ATOM 1914 N ARG A 303 226.630 85.611 211.878 1.00 73.53 N ATOM1915 CA ARG A 303 225.526 84.913 212.532 1.00 61.85 C ATOM 1916 C ARG A303 224.162 85.201 211.898 1.00 74.05 C ATOM 1917 O ARG A 303 223.37084.286 211.695 1.00 79.80 O ATOM 1918 CB ARG A 303 225.461 85.279214.009 1.00 61.93 C ATOM 1919 CG ARG A 303 226.666 84.917 214.852 1.0078.75 C ATOM 1920 CD ARG A 303 226.402 85.392 216.284 1.00 71.93 C ATOM1921 NE ARG A 303 227.608 85.725 217.038 1.00 101.37 N ATOM 1922 CZ ARGA 303 228.488 84.827 217.457 1.00 93.09 C ATOM 1923 NH1 ARG A 303228.286 83.540 217.191 1.00 90.11 N ATOM 1924 NH2 ARG A 303 229.56185.211 218.140 1.00 76.11 N ATOM 1925 N ASN A 304 223.865 86.458 211.5961.00 61.15 N ATOM 1926 CA ASN A 304 222.575 86.775 210.982 1.00 77.76 CATOM 1927 C ASN A 304 222.517 86.277 209.535 1.00 74.95 C ATOM 1928 OASN A 304 221.449 86.208 208.929 1.00 67.98 O ATOM 1929 CB ASN A 304222.312 88.291 211.016 1.00 83.64 C ATOM 1930 CG ASN A 304 222.00088.806 212.421 1.00 112.22 C ATOM 1931 OD1 ASN A 304 222.399 89.919212.802 1.00 84.27 O ATOM 1932 ND2 ASN A 304 221.269 88.004 213.197 1.0069.02 N ATOM 1933 N LEU A 305 223.671 85.943 208.973 1.00 63.99 N ATOM1934 CA LEU A 305 223.711 85.458 207.605 1.00 54.76 C ATOM 1935 C LEU A305 223.590 83.952 207.666 1.00 73.10 C ATOM 1936 O LEU A 305 222.92983.313 206.841 1.00 58.22 O ATOM 1937 CB LEU A 305 225.032 85.860206.948 1.00 52.05 C ATOM 1938 CG LEU A 305 225.127 87.316 206.481 1.0058.75 C ATOM 1939 CD1 LEU A 305 226.511 87.621 205.932 1.00 69.80 C ATOM1940 CD2 LEU A 305 224.075 87.560 205.421 1.00 47.92 C ATOM 1941 N LEU A306 224.240 83.401 208.681 1.00 57.81 N ATOM 1942 CA LEU A 306 224.25681.975 208.915 1.00 59.22 C ATOM 1943 C LEU A 306 223.087 81.525 209.7791.00 70.17 C ATOM 1944 O LEU A 306 223.205 80.586 210.565 1.00 86.15 OATOM 1945 CB LEU A 306 225.581 81.582 209.559 1.00 54.83 C ATOM 1946 CGLEU A 306 226.752 81.718 208.599 1.00 56.28 C ATOM 1947 CD1 LEU A 306228.035 81.262 209.237 1.00 46.67 C ATOM 1948 CD2 LEU A 306 226.44580.883 207.385 1.00 50.52 C ATOM 1949 N LYS A 307 221.960 82.212 209.6481.00 70.52 N ATOM 1950 CA LYS A 307 220.772 81.832 210.394 1.00 64.84 CATOM 1951 C LYS A 307 220.251 80.593 209.676 1.00 65.05 C ATOM 1952 OLYS A 307 220.153 80.570 208.449 1.00 73.22 O ATOM 1953 CB LYS A 307219.720 82.930 210.337 1.00 60.26 C ATOM 1954 CG LYS A 307 220.13784.247 210.952 1.00 76.35 C ATOM 1955 CD LYS A 307 219.620 84.411212.371 1.00 75.87 C ATOM 1956 CE LYS A 307 220.487 83.696 213.389 1.0092.33 C ATOM 1957 NZ LYS A 307 219.963 83.886 214.773 1.00 94.73 N ATOM1958 N THR A 308 219.925 79.558 210.439 1.00 87.36 N ATOM 1959 CA THR A308 219.432 78.314 209.864 1.00 86.54 C ATOM 1960 C THR A 308 218.05778.489 209.219 1.00 80.07 C ATOM 1961 O THR A 308 217.722 77.809 208.2481.00 80.48 O ATOM 1962 CB THR A 308 219.382 77.198 210.945 1.00 80.31 CATOM 1963 OG1 THR A 308 220.615 76.457 210.929 1.00 85.50 O ATOM 1964CG2 THR A 308 218.211 76.265 210.701 1.00 108.80 C ATOM 1965 N GLU A 309217.264 79.407 209.755 1.00 86.56 N ATOM 1966 CA GLU A 309 215.93479.647 209.217 1.00 80.34 C ATOM 1967 C GLU A 309 215.964 80.820 208.2531.00 80.14 C ATOM 1968 O GLU A 309 216.353 81.933 208.614 1.00 72.43 OATOM 1969 CB GLU A 309 214.937 79.945 210.345 1.00 79.03 C ATOM 1970 CGGLU A 309 213.461 79.716 209.976 1.00 107.32 C ATOM 1971 CD GLU A 309213.028 78.264 210.160 1.00 128.86 C ATOM 1972 OE1 GLU A 309 213.35477.691 211.227 1.00 100.32 O ATOM 1973 OE2 GLU A 309 212.364 77.699209.252 1.00 91.75 O ATOM 1974 N PRO A 310 215.554 80.585 207.002 1.0069.20 N ATOM 1975 CA PRO A 310 215.536 81.644 205.988 1.00 75.56 C ATOM1976 C PRO A 310 214.955 82.953 206.540 1.00 73.13 C ATOM 1977 O PRO A310 215.697 83.886 206.848 1.00 70.20 O ATOM 1978 CB PRO A 310 214.67581.041 204.888 1.00 58.28 C ATOM 1979 CG PRO A 310 215.049 79.598204.957 1.00 61.31 C ATOM 1980 CD PRO A 310 215.073 79.312 206.442 1.0064.66 C ATOM 1981 N THR A 311 213.633 83.004 206.683 1.00 57.71 N ATOM1982 CA THR A 311 212.952 84.188 207.194 1.00 63.44 C ATOM 1983 C THR A311 213.671 84.899 208.338 1.00 65.40 C ATOM 1984 O THR A 311 213.42486.078 208.585 1.00 70.08 O ATOM 1985 CB THR A 311 211.552 83.839207.666 1.00 52.68 C ATOM 1986 OG1 THR A 311 211.650 82.936 208.763 1.0076.00 O ATOM 1987 CG2 THR A 311 210.781 83.179 206.574 1.00 44.79 C ATOM1988 N GLN A 312 214.551 84.198 209.043 1.00 61.23 N ATOM 1989 CA GLN A312 215.271 84.831 210.145 1.00 58.30 C ATOM 1990 C GLN A 312 216.52785.518 209.629 1.00 63.30 C ATOM 1991 O GLN A 312 216.995 86.509 210.1891.00 65.70 O ATOM 1992 CB GLN A 312 215.675 83.798 211.202 1.00 78.77 CATOM 1993 CG GLN A 312 215.203 84.120 212.625 1.00 101.21 C ATOM 1994 CDGLN A 312 215.952 83.321 213.691 1.00 105.49 C ATOM 1995 OE1 GLN A 312217.112 83.612 214.004 1.00 110.73 O ATOM 1996 NE2 GLN A 312 215.29482.305 214.243 1.00 82.13 N ATOM 1997 N ARG A 313 217.057 84.974 208.5451.00 75.75 N ATOM 1998 CA ARG A 313 218.278 85.463 207.924 1.00 58.86 CATOM 1999 C ARG A 313 218.184 86.894 207.391 1.00 51.81 C ATOM 2000 OARG A 313 217.125 87.341 206.952 1.00 85.05 O ATOM 2001 CB ARG A 313218.663 84.495 206.807 1.00 52.47 C ATOM 2002 CG ARG A 313 220.13184.401 206.516 1.00 74.86 C ATOM 2003 CD ARG A 313 220.355 83.299205.506 1.00 84.86 C ATOM 2004 NE ARG A 313 219.851 82.018 205.984 1.0053.52 N ATOM 2005 CZ ARG A 313 219.467 81.032 205.186 1.00 62.95 C ATOM2006 NH1 ARG A 313 219.528 81.192 203.874 1.00 85.30 N ATOM 2007 NH2 ARGA 313 219.039 79.888 205.697 1.00 49.87 N ATOM 2008 N MET A 314 219.31887.590 207.430 1.00 75.49 N ATOM 2009 CA MET A 314 219.450 88.981206.980 1.00 62.90 C ATOM 2010 C MET A 314 219.115 89.160 205.496 1.0068.20 C ATOM 2011 O MET A 314 219.267 88.229 204.694 1.00 78.91 O ATOM2012 CB MET A 314 220.888 89.463 207.248 1.00 79.70 C ATOM 2013 CG MET A314 221.145 90.951 206.994 1.00 74.01 C ATOM 2014 SD MET A 314 222.87391.447 207.330 1.00 84.44 S ATOM 2015 CE MET A 314 222.742 92.071208.957 1.00 104.75 C ATOM 2016 N THR A 315 218.660 90.357 205.135 1.0063.73 N ATOM 2017 CA THR A 315 218.319 90.652 203.750 1.00 64.98 C ATOM2018 C THR A 315 219.470 91.347 203.030 1.00 68.09 C ATOM 2019 O THR A315 220.317 91.969 203.658 1.00 63.59 O ATOM 2020 CB THR A 315 217.07591.545 203.661 1.00 62.15 C ATOM 2021 OG1 THR A 315 217.322 92.813204.286 1.00 58.05 O ATOM 2022 CG2 THR A 315 215.929 90.868 204.338 1.0061.97 C ATOM 2023 N ILE A 316 219.498 91.236 201.707 1.00 65.76 N ATOM2024 CA ILE A 316 220.566 91.854 200.935 1.00 66.14 C ATOM 2025 C ILE A316 220.564 93.351 201.167 1.00 71.26 C ATOM 2026 O ILE A 316 221.61893.981 201.192 1.00 67.36 O ATOM 2027 CB ILE A 316 220.417 91.583199.426 1.00 60.35 C ATOM 2028 CG1 ILE A 316 221.714 91.937 198.710 1.0052.24 C ATOM 2029 CG2 ILE A 316 219.314 92.435 198.854 1.00 56.27 C ATOM2030 CD1 ILE A 316 222.942 91.410 199.405 1.00 55.83 C ATOM 2031 N THR A317 219.381 93.926 201.346 1.00 60.50 N ATOM 2032 CA THR A 317 219.30195.356 201.591 1.00 58.94 C ATOM 2033 C THR A 317 219.858 95.658 202.9641.00 61.73 C ATOM 2034 O THR A 317 220.533 96.659 203.161 1.00 69.91 OATOM 2035 CB THR A 317 217.865 95.875 201.554 1.00 68.58 C ATOM 2036 OG1THR A 317 217.301 95.636 200.259 1.00 67.56 O ATOM 2037 CG2 THR A 317217.844 97.364 201.860 1.00 50.05 C ATOM 2038 N GLU A 318 219.577 94.794203.923 1.00 54.23 N ATOM 2039 CA GLU A 318 220.082 95.027 205.266 1.0069.88 C ATOM 2040 C GLU A 318 221.586 94.796 205.348 1.00 72.99 C ATOM2041 O GLU A 318 222.262 95.329 206.221 1.00 64.78 O ATOM 2042 CB GLU A318 219.336 94.138 206.269 1.00 82.03 C ATOM 2043 CG GLU A 318 217.90394.596 206.496 1.00 64.99 C ATOM 2044 CD GLU A 318 217.152 93.742207.486 1.00 85.92 C ATOM 2045 OE1 GLU A 318 216.415 94.327 208.315 1.0091.71 O ATOM 2046 OE2 GLU A 318 217.288 92.497 207.424 1.00 88.17 O ATOM2047 N PHE A 319 222.103 94.005 204.421 1.00 75.36 N ATOM 2048 CA PHE A319 223.519 93.700 204.380 1.00 69.31 C ATOM 2049 C PHE A 319 224.27994.883 203.800 1.00 79.85 C ATOM 2050 O PHE A 319 225.281 95.324 204.3591.00 86.43 O ATOM 2051 CB PHE A 319 223.744 92.455 203.523 1.00 78.91 CATOM 2052 CG PHE A 319 225.193 92.144 203.250 1.00 82.05 C ATOM 2053 CD1PHE A 319 225.978 91.538 204.210 1.00 64.69 C ATOM 2054 CD2 PHE A 319225.757 92.432 202.011 1.00 68.65 C ATOM 2055 CE1 PHE A 319 227.28791.219 203.940 1.00 55.30 C ATOM 2056 CE2 PHE A 319 227.060 92.114201.744 1.00 75.06 C ATOM 2057 CZ PHE A 319 227.826 91.506 202.711 1.0057.56 C ATOM 2058 N MET A 320 223.805 95.404 202.679 1.00 64.36 N ATOM2059 CA MET A 320 224.489 96.527 202.066 1.00 69.16 C ATOM 2060 C MET A320 224.525 97.753 202.973 1.00 78.25 C ATOM 2061 O MET A 320 225.30498.673 202.745 1.00 86.13 O ATOM 2062 CB MET A 320 223.848 96.889200.725 1.00 73.27 C ATOM 2063 CG MET A 320 224.120 95.877 199.635 1.0072.98 C ATOM 2064 SD MET A 320 225.832 95.315 199.691 1.00 87.32 S ATOM2065 CE MET A 320 226.586 96.413 198.528 1.00 70.20 C ATOM 2066 N ASN A321 223.695 97.772 204.006 1.00 77.28 N ATOM 2067 CA ASN A 321 223.69198.907 204.912 1.00 79.74 C ATOM 2068 C ASN A 321 224.489 98.692 206.1741.00 76.50 C ATOM 2069 O ASN A 321 224.868 99.651 206.836 1.00 95.86 OATOM 2070 CB ASN A 321 222.271 99.296 205.267 1.00 79.55 C ATOM 2071 CGASN A 321 221.783 100.428 204.427 1.00 104.33 C ATOM 2072 OD1 ASN A 321222.191 101.578 204.622 1.00 115.09 O ATOM 2073 ND2 ASN A 321 220.926100.121 203.456 1.00 119.81 N ATOM 2074 N HIS A 322 224.744 97.439206.521 1.00 82.50 N ATOM 2075 CA HIS A 322 225.527 97.179 207.707 1.0080.78 C ATOM 2076 C HIS A 322 226.837 97.948 207.555 1.00 84.39 C ATOM2077 O HIS A 322 227.483 97.895 206.506 1.00 83.28 O ATOM 2078 CB HIS A322 225.812 95.691 207.851 1.00 75.27 C ATOM 2079 CG HIS A 322 226.69095.370 209.013 1.00 76.67 C ATOM 2080 ND1 HIS A 322 227.927 95.950209.189 1.00 84.96 N ATOM 2081 CD2 HIS A 322 226.504 94.548 210.070 1.0067.42 C ATOM 2082 CE1 HIS A 322 228.464 95.502 210.308 1.00 85.58 C ATOM2083 NE2 HIS A 322 227.622 94.649 210.862 1.00 86.63 N ATOM 2084 N PRO A323 227.234 98.686 208.603 1.00 91.61 N ATOM 2085 CA PRO A 323 228.44799.501 208.680 1.00 84.82 C ATOM 2086 C PRO A 323 229.697 98.867 208.0871.00 79.23 C ATOM 2087 O PRO A 323 230.318 99.436 207.193 1.00 85.59 OATOM 2088 CB PRO A 323 228.567 99.772 210.170 1.00 74.59 C ATOM 2089 CGPRO A 323 227.135 99.952 210.553 1.00 86.91 C ATOM 2090 CD PRO A 323226.484 98.767 209.868 1.00 92.14 C ATOM 2091 N TRP A 324 230.066 97.692208.578 1.00 81.53 N ATOM 2092 CA TRP A 324 231.247 97.002 208.070 1.0080.47 C ATOM 2093 C TRP A 324 231.260 97.039 206.541 1.00 76.75 C ATOM2094 O TRP A 324 232.288 97.293 205.917 1.00 63.50 O ATOM 2095 CB TRP A324 231.225 95.553 208.537 1.00 79.62 C ATOM 2096 CG TRP A 324 232.54994.881 208.546 1.00 68.42 C ATOM 2097 CD1 TRP A 324 233.579 95.118209.408 1.00 97.23 C ATOM 2098 CD2 TRP A 324 232.973 93.806 207.704 1.0091.77 C ATOM 2099 NE1 TRP A 324 234.614 94.254 209.162 1.00 96.32 N ATOM2100 CE2 TRP A 324 234.268 93.437 208.120 1.00 90.15 C ATOM 2101 CE3 TRPA 324 232.382 93.118 206.639 1.00 85.73 C ATOM 2102 CZ2 TRP A 324234.979 92.411 207.515 1.00 55.96 C ATOM 2103 CZ3 TRP A 324 233.09192.099 206.038 1.00 78.54 C ATOM 2104 CH2 TRP A 324 234.375 91.755206.475 1.00 83.38 C ATOM 2105 N ILE A 325 230.099 96.785 205.952 1.0061.98 N ATOM 2106 CA ILE A 325 229.956 96.764 204.513 1.00 63.34 C ATOM2107 C ILE A 325 229.832 98.156 203.920 1.00 81.46 C ATOM 2108 O ILE A325 230.518 98.483 202.956 1.00 101.97 O ATOM 2109 CB ILE A 325 228.72395.941 204.092 1.00 64.13 C ATOM 2110 CG1 ILE A 325 229.156 94.621203.470 1.00 57.81 C ATOM 2111 CG2 ILE A 325 227.909 96.703 203.076 1.0079.69 C ATOM 2112 CD1 ILE A 325 230.005 93.796 204.357 1.00 99.33 C ATOM2113 N MET A 326 228.971 98.986 204.497 1.00 86.84 N ATOM 2114 CA MET A326 228.766 100.327 203.962 1.00 97.80 C ATOM 2115 C MET A 326 229.941101.291 204.108 1.00 88.82 C ATOM 2116 O MET A 326 230.023 102.271203.375 1.00 96.42 O ATOM 2117 CB MET A 326 227.509 100.951 204.573 1.0099.22 C ATOM 2118 CG MET A 326 227.134 102.287 203.957 1.00 109.19 CATOM 2119 SD MET A 326 225.425 102.771 204.309 1.00 131.16 S ATOM 2120CE MET A 326 224.679 102.571 202.683 1.00 123.49 C ATOM 2121 N GLN A 327230.847 101.016 205.042 1.00 99.42 N ATOM 2122 CA GLN A 327 232.005101.880 205.253 1.00 100.47 C ATOM 2123 C GLN A 327 233.330 101.120205.290 1.00 109.31 C ATOM 2124 O GLN A 327 234.060 101.184 206.279 1.00116.79 O ATOM 2125 CB GLN A 327 231.856 102.668 206.557 1.00 90.31 CATOM 2126 CG GLN A 327 230.780 103.743 206.538 1.00 124.03 C ATOM 2127CD GLN A 327 230.929 104.713 205.370 1.00 165.27 C ATOM 2128 OE1 GLN A327 232.047 105.051 204.958 1.00 160.87 O ATOM 2129 NE2 GLN A 327229.795 105.178 204.841 1.00 148.79 N ATOM 2130 N SER A 328 233.644100.406 204.215 1.00 109.39 N ATOM 2131 CA SER A 328 234.888 99.643204.157 1.00 117.56 C ATOM 2132 C SER A 328 236.084 100.539 204.457 1.00126.63 C ATOM 2133 O SER A 328 236.892 100.239 205.332 1.00 122.39 OATOM 2134 CB SER A 328 235.055 99.008 202.777 1.00 106.48 C ATOM 2135 OGSER A 328 233.976 98.139 202.490 1.00 101.32 O ATOM 2136 N THR A 329236.189 101.642 203.725 1.00 147.22 N ATOM 2137 CA THR A 329 237.283102.582 203.919 1.00 153.16 C ATOM 2138 C THR A 329 237.668 102.713205.393 1.00 151.36 C ATOM 2139 O THR A 329 238.849 102.680 205.736 1.00165.48 O ATOM 2140 CB THR A 329 236.918 103.986 203.365 1.00 159.02 CATOM 2141 OG1 THR A 329 237.929 104.930 203.748 1.00 164.35 O ATOM 2142CG2 THR A 329 235.553 104.443 203.893 1.00 171.23 C ATOM 2143 N LYS A330 236.670 102.840 206.262 1.00 138.67 N ATOM 2144 CA LYS A 330 236.916102.994 207.691 1.00 128.38 C ATOM 2145 C LYS A 330 236.937 101.670208.457 1.00 134.38 C ATOM 2146 O LYS A 330 236.425 101.586 209.580 1.00138.61 O ATOM 2147 CB LYS A 330 235.867 103.931 208.298 1.00 134.91 CATOM 2148 N VAL A 331 237.526 100.637 207.860 1.00 122.49 N ATOM 2149 CAVAL A 331 237.608 99.340 208.531 1.00 120.28 C ATOM 2150 C VAL A 331239.066 98.890 208.689 1.00 111.19 C ATOM 2151 O VAL A 331 239.85098.899 207.739 1.00 85.41 O ATOM 2152 CB VAL A 331 236.770 98.239207.785 1.00 120.15 C ATOM 2153 CG1 VAL A 331 237.342 97.971 206.4221.00 125.69 C ATOM 2154 CG2 VAL A 331 236.740 96.946 208.595 1.00 124.20C ATOM 2155 N PRO A 332 239.433 98.496 209.916 1.00 102.76 N ATOM 2156CA PRO A 332 240.737 98.021 210.385 1.00 105.52 C ATOM 2157 C PRO A 332241.528 97.215 209.363 1.00 112.97 C ATOM 2158 O PRO A 332 241.01696.262 208.779 1.00 111.93 O ATOM 2159 CB PRO A 332 240.369 97.193211.609 1.00 105.70 C ATOM 2160 CG PRO A 332 239.247 97.970 212.184 1.00122.25 C ATOM 2161 CD PRO A 332 238.418 98.332 210.971 1.00 100.61 CATOM 2162 N GLN A 333 242.782 97.604 209.154 1.00 109.39 N ATOM 2163 CAGLN A 333 243.645 96.908 208.206 1.00 106.56 C ATOM 2164 C GLN A 333244.265 95.714 208.913 1.00 99.10 C ATOM 2165 O GLN A 333 245.347 95.251208.554 1.00 97.72 O ATOM 2166 CB GLN A 333 244.744 97.842 207.696 1.00117.29 C ATOM 2167 CG GLN A 333 244.224 99.042 206.922 1.00 121.73 CATOM 2168 CD GLN A 333 244.771 99.095 205.510 1.00 144.41 C ATOM 2169OE1 GLN A 333 244.442 99.996 204.735 1.00 153.63 O ATOM 2170 NE2 GLN A333 245.613 98.124 205.165 1.00 138.94 N ATOM 2171 N THR A 334 243.56595.229 209.931 1.00 88.60 N ATOM 2172 CA THR A 334 244.023 94.081210.693 1.00 112.85 C ATOM 2173 C THR A 334 244.310 92.878 209.797 1.00123.95 C ATOM 2174 O THR A 334 243.438 92.392 209.086 1.00 128.29 O ATOM2175 CB THR A 334 242.989 93.668 211.738 1.00 105.19 C ATOM 2176 OG1 THRA 334 243.011 92.243 211.887 1.00 102.93 O ATOM 2177 CG2 THR A 334241.607 94.120 211.315 1.00 106.97 C ATOM 2178 N PRO A 335 245.54992.372 209.832 1.00 124.58 N ATOM 2179 CA PRO A 335 245.911 91.224209.005 1.00 124.62 C ATOM 2180 C PRO A 335 245.058 89.999 209.317 1.00119.67 C ATOM 2181 O PRO A 335 244.513 89.878 210.416 1.00 108.06 O ATOM2182 CB PRO A 335 247.383 91.002 209.358 1.00 134.66 C ATOM 2183 CG PROA 335 247.857 92.390 209.673 1.00 120.03 C ATOM 2184 CD PRO A 335246.730 92.881 210.547 1.00 120.76 C ATOM 2185 N LEU A 336 244.92689.110 208.333 1.00 110.96 N ATOM 2186 CA LEU A 336 244.175 87.867208.499 1.00 98.45 C ATOM 2187 C LEU A 336 245.087 86.726 208.066 1.0087.77 C ATOM 2188 O LEU A 336 245.851 86.877 207.117 1.00 92.64 O ATOM2189 CB LEU A 336 242.909 87.852 207.632 1.00 95.92 C ATOM 2190 CG LEU A336 241.694 88.725 207.963 1.00 81.15 C ATOM 2191 CD1 LEU A 336 241.64188.972 209.461 1.00 94.01 C ATOM 2192 CD2 LEU A 336 241.773 90.034207.215 1.00 82.79 C ATOM 2193 N HIS A 337 244.999 85.595 208.762 1.0086.05 N ATOM 2194 CA HIS A 337 245.813 84.410 208.469 1.00 100.05 C ATOM2195 C HIS A 337 245.401 83.709 207.170 1.00 103.25 C ATOM 2196 O HIS A337 246.075 82.782 206.712 1.00 110.52 O ATOM 2197 CB HIS A 337 245.68783.390 209.610 1.00 118.04 C ATOM 2198 CG HIS A 337 246.205 83.874210.930 1.00 151.49 C ATOM 2199 ND1 HIS A 337 246.241 85.210 211.2711.00 169.76 N ATOM 2200 CD2 HIS A 337 246.675 83.198 212.010 1.00 160.51C ATOM 2201 CE1 HIS A 337 246.713 85.337 212.502 1.00 129.99 C ATOM 2202NE2 HIS A 337 246.984 84.132 212.971 1.00 150.26 N ATOM 2203 N THR A 338244.294 84.167 206.589 1.00 111.23 N ATOM 2204 CA THR A 338 243.71383.590 205.375 1.00 105.16 C ATOM 2205 C THR A 338 244.649 82.822204.440 1.00 106.58 C ATOM 2206 O THR A 338 244.708 81.589 204.504 1.00102.89 O ATOM 2207 CB THR A 338 242.957 84.658 204.557 1.00 100.36 CATOM 2208 OG1 THR A 338 242.218 85.494 205.451 1.00 103.82 O ATOM 2209CG2 THR A 338 241.961 83.993 203.605 1.00 97.07 C ATOM 2210 N SER A 339245.370 83.532 203.573 1.00 100.67 N ATOM 2211 CA SER A 339 246.27682.877 202.622 1.00 106.75 C ATOM 2212 C SER A 339 246.957 81.641203.202 1.00 117.17 C ATOM 2213 O SER A 339 246.862 80.555 202.634 1.00126.71 O ATOM 2214 CB SER A 339 247.341 83.855 202.137 1.00 100.87 CATOM 2215 OG SER A 339 246.771 84.880 201.348 1.00 116.32 O ATOM 2216 NARG A 340 247.643 81.811 204.329 1.00 108.64 N ATOM 2217 CA ARG A 340248.325 80.697 204.971 1.00 101.87 C ATOM 2218 C ARG A 340 247.39179.508 205.121 1.00 94.03 C ATOM 2219 O ARG A 340 247.554 78.480 204.4541.00 80.82 O ATOM 2220 CB ARG A 340 248.848 81.112 206.347 1.00 117.73 CATOM 2221 CG ARG A 340 250.041 82.055 206.292 1.00 162.36 C ATOM 2222 CDARG A 340 249.770 83.353 207.068 1.00 208.75 C ATOM 2223 NE ARG A 340249.607 83.107 208.504 1.00 216.90 N ATOM 2224 CZ ARG A 340 249.37884.053 209.416 1.00 203.42 C ATOM 2225 NH1 ARG A 340 249.279 85.330209.055 1.00 186.56 N ATOM 2226 NH2 ARG A 340 249.255 83.719 210.6961.00 191.24 N ATOM 2227 N VAL A 341 246.412 79.653 206.004 1.00 84.97 NATOM 2228 CA VAL A 341 245.455 78.585 206.239 1.00 88.40 C ATOM 2229 CVAL A 341 244.913 78.128 204.891 1.00 93.56 C ATOM 2230 O VAL A 341244.917 76.939 204.566 1.00 86.14 O ATOM 2231 CB VAL A 341 244.26979.077 207.078 1.00 86.60 C ATOM 2232 CG1 VAL A 341 243.692 77.928207.866 1.00 93.99 C ATOM 2233 CG2 VAL A 341 244.700 80.214 207.975 1.0087.95 C ATOM 2234 N LEU A 342 244.463 79.101 204.107 1.00 98.12 N ATOM2235 CA LEU A 342 243.887 78.847 202.798 1.00 97.33 C ATOM 2236 C LEU A342 244.771 77.929 201.976 1.00 103.21 C ATOM 2237 O LEU A 342 244.28077.134 201.181 1.00 108.03 O ATOM 2238 CB LEU A 342 243.666 80.171202.065 1.00 101.09 C ATOM 2239 CG LEU A 342 242.513 80.212 201.065 1.00102.01 C ATOM 2240 CD1 LEU A 342 241.211 79.865 201.761 1.00 89.86 CATOM 2241 CD2 LEU A 342 242.428 81.595 200.463 1.00 76.62 C ATOM 2242 NLYS A 343 246.078 78.031 202.177 1.00 118.91 N ATOM 2243 CA LYS A 343246.980 77.154 201.447 1.00 128.99 C ATOM 2244 C LYS A 343 247.21175.800 202.144 1.00 129.81 C ATOM 2245 O LYS A 343 247.730 74.859201.559 1.00 124.36 O ATOM 2246 CB LYS A 343 248.308 77.894 201.320 1.00131.78 C ATOM 2247 CG LYS A 343 249.233 77.265 200.274 1.00 133.47 CATOM 2248 CD LYS A 343 250.528 78.061 200.106 1.00 161.36 C ATOM 2249 CELYS A 343 251.591 77.288 199.318 1.00 189.16 C ATOM 2250 NZ LYS A 343252.882 77.968 199.425 1.00 166.57 N ATOM 2251 N GLU A 344 246.84275.714 203.442 1.00 128.07 N ATOM 2252 CA GLU A 344 247.254 74.549204.240 1.00 135.55 C ATOM 2253 C GLU A 344 246.429 73.272 203.985 1.00137.12 C ATOM 2254 O GLU A 344 246.915 72.156 204.102 1.00 147.11 O ATOM2255 CB GLU A 344 247.196 74.935 205.720 1.00 144.51 C ATOM 2256 CG GLUA 344 248.480 74.589 206.485 1.00 182.52 C ATOM 2257 CD GLU A 344249.414 75.782 206.484 1.00 205.75 C ATOM 2258 OE1 GLU A 344 249.25276.650 207.336 1.00 195.59 O ATOM 2259 OE2 GLU A 344 250.270 75.861205.607 1.00 217.38 O ATOM 2260 N ASP A 345 245.127 73.456 203.702 1.00134.97 N ATOM 2261 CA ASP A 345 244.310 72.298 203.321 1.00 153.39 CATOM 2262 C ASP A 345 243.429 72.611 202.107 1.00 164.28 C ATOM 2263 OASP A 345 243.779 73.413 201.250 1.00 177.09 O ATOM 2264 CB ASP A 345243.435 71.892 204.514 1.00 147.36 C ATOM 2265 CG ASP A 345 242.85170.495 204.291 1.00 174.56 C ATOM 2266 OD1 ASP A 345 243.455 69.731203.533 1.00 191.84 O ATOM 2267 OD2 ASP A 345 241.820 70.182 204.8831.00 169.86 O TER 2267 ASP A 345 ATOM 1 N LEU C 4 242.399 73.184 199.5391.00 15.00 ATOM 2 CA LEU C 4 240.997 73.382 199.193 1.00 15.00 ATOM 3 CBLEU C 4 240.688 74.874 199.065 1.00 15.00 ATOM 4 CG LEU C 4 239.46275.245 198.226 1.00 15.00 ATOM 5 CD1 LEU C 4 238.302 75.613 199.137 1.0015.00 ATOM 6 CD2 LEU C 4 239.801 76.397 197.295 1.00 15.00 ATOM 7 C LEUC 4 240.650 72.668 197.890 1.00 15.00 ATOM 8 O LEU C 4 241.075 72.982196.773 1.00 22.73 ATOM 9 N GLN C 5 239.765 71.671 197.981 1.00 15.00ATOM 10 CA GLN C 5 239.268 70.862 196.875 1.00 15.00 ATOM 11 CB GLN C 5238.690 69.547 197.399 1.00 15.00 ATOM 12 CG GLN C 5 238.232 69.602198.847 1.00 15.00 ATOM 13 CD GLN C 5 238.424 68.283 199.571 1.00 15.00ATOM 14 OE1 GLN C 5 237.494 67.758 200.184 1.00 15.00 ATOM 15 NE2 GLN C5 239.634 67.743 199.504 1.00 15.00 ATOM 16 C GLN C 5 238.205 71.615196.082 1.00 15.00 ATOM 17 O GLN C 5 237.316 72.263 196.691 1.00 15.04ATOM 18 N ARG C 6 238.236 71.564 194.739 1.00 18.35 N ATOM 19 CA ARG C 6237.243 72.209 193.873 1.00 18.05 C ATOM 20 C ARG C 6 235.863 71.634194.126 1.00 17.37 C ATOM 21 O ARG C 6 235.711 70.424 194.303 1.00 16.29O ATOM 22 CB ARG C 6 237.578 72.015 192.398 1.00 20.66 C ATOM 23 CG ARGC 6 238.842 72.678 191.953 1.00 23.47 C ATOM 24 CD ARG C 6 238.84072.882 190.445 1.00 23.04 C ATOM 25 NE ARG C 6 237.880 73.892 189.9811.00 22.74 N ATOM 26 CZ ARG C 6 237.975 75.204 190.210 1.00 20.22 C ATOM27 NH1 ARG C 6 238.987 75.699 190.913 1.00 19.12 N ATOM 28 NH2 ARG C 6237.069 76.031 189.710 1.00 20.28 N ATOM 29 N GLN C 7 234.864 72.520194.186 1.00 15.00 ATOM 30 CA GLN C 7 233.488 72.105 194.427 1.00 15.00ATOM 31 CB GLN C 7 232.904 72.869 195.617 1.00 15.00 ATOM 32 CG GLN C 7233.852 72.996 196.798 1.00 15.00 ATOM 33 CD GLN C 7 233.258 73.799197.940 1.00 15.00 ATOM 34 OE1 GLN C 7 232.067 74.116 197.937 1.00 15.00ATOM 35 NE2 GLN C 7 234.086 74.132 198.923 1.00 15.00 ATOM 36 C GLN C 7232.623 72.336 193.191 1.00 15.00 ATOM 37 O GLN C 7 232.724 73.384192.538 1.00 20.09 ATOM 38 N LEU C 8 231.805 71.324 192.833 1.00 15.00ATOM 39 CA LEU C 8 230.922 71.415 191.677 1.00 15.00 ATOM 40 CB LEU C 8231.160 70.233 190.734 1.00 15.00 ATOM 41 CG LEU C 8 231.208 70.558189.238 1.00 15.00 ATOM 42 CD1 LEU C 8 232.592 70.252 188.686 1.00 15.00ATOM 43 CD2 LEU C 8 230.147 69.759 188.500 1.00 15.00 ATOM 44 C LEU C 8229.459 71.446 192.108 1.00 15.00 ATOM 45 O LEU C 8 229.098 70.977193.190 1.00 13.73 ATOM 46 N SER C 9 228.611 72.005 191.242 1.00 13.08 NATOM 47 CA SER C 9 227.183 72.104 191.526 1.00 12.87 C ATOM 48 C SER C 9226.402 70.843 191.151 1.00 13.16 C ATOM 49 O SER C 9 226.930 69.943190.490 1.00 14.95 O ATOM 50 CB SER C 9 226.569 73.323 190.823 1.0014.61 C ATOM 51 OG SER C 9 226.326 73.072 189.444 1.00 14.77 O ATOM 52 NILE C 10 225.174 70.780 191.613 1.00 15.00 ATOM 53 CA ILE C 10 224.24969.694 191.311 1.00 15.00 ATOM 54 CB ILE C 10 223.837 68.939 192.5911.00 15.00 ATOM 55 CG2 ILE C 10 225.025 68.817 193.532 1.00 15.00 ATOM56 CG1 ILE C 10 222.681 69.664 193.280 1.00 15.00 ATOM 57 CD1 ILE C 10222.397 69.168 194.682 1.00 15.00 ATOM 58 C ILE C 10 222.992 70.220190.626 1.00 15.00 ATOM 59 O ILE C 10 222.755 71.452 190.654 1.00 12.25ATOM 60 N ALA C 11 222.224 69.342 190.029 1.00 14.49 N ATOM 61 CA ALA C11 220.983 69.676 189.342 1.00 16.79 C ATOM 62 C ALA C 11 220.122 68.421189.365 1.00 19.44 C ATOM 63 O ALA C 11 220.552 67.360 188.901 1.0019.58 O ATOM 64 CB ALA C 11 221.252 70.125 187.902 1.00 15.87 C TER 64ALA C 11 END

TABLE 2 Pairs of contacting atoms in the MAPKAP kinase-2/peptide complexAtom in activated MAPKAP kinase-2 Atom in peptide Distance ILE74 CD1SER9 OG 2.90275 GLU145 OE1 ARG6 NH2 1.94903 LYS188 NZ SER9 CB 2.48016GLU190 CD GLN7 O 3.33866 PHE210 CE2 SER9 OG 2.78278 PHE210 CZ ILE10 O2.37786 CYS224 SG ALA11 CA 3.36808 TYR225 O SER9 CA 3.25655 TYR225 OILE10 N 2.88016 THR 226 OG1 GLN 7 OE1 3.42713 PRO 227 CD LEU 8 O 3.44686TYR 228 CB GLN 7 OE1 3.02662 TYR 229 CE1 GLN 7 OE1 3.34283 ASP 345 O LEU4 N 2.21006

RNA interference (RNAi) and recombinant DNA. siRNA duplexes consistingof twenty-one base pairs with a two-base deoxynucleotide overhang werepurchased from Dharmacon Research. Cells were transfected with siRNAsusing oligofectamine (Invitrogen) according to the manufacturer'sinstructions. Cells were typically harvested for further experimentsafter forty-eight hours. U2OS cells stably expressing shRNA constructswere generated by lentiviral gene transfer. The RNAi hairpins werecloned into the multiple cloning site of the lentiviral transfer vectorpLentiLox-3.7puro or -3.7GFP. Amphotropic VSV-G pseudotyped lentiviruswas used for all infections in a BL2+ facility. All transfer andpackaging constructs were a kind gift from C.P. Dillon, (MIT). Targetedcells were selected in 8 μg/ml puromycin for four days. Sequences usedfor RNAi were: luciferase (shRNA), 5′pTGA CCA GGC ATT CAC AGA AAT TCAAGA GAT TTC TGT GAA TGC CTG GTC TTT TTT C-3′ (SEQ ID NO: 24); hMAPKAPkinase-2 (shRNA), 5′-pTTG ACC ATC ACC GAG TTT ATT TCA AGA GAA TA AAC TCGGTG ATG GTC ATT TTT TC-3′ (SEQ ID NO: 25); mMAPKAP kinase-2 (shRNA),5′-pTCG ATG CGT GTT GAC TAT GAT TCA AGA GAT CAT AGT CAA CAC GCA TCG TTTTTT C-3′ (SEQ ID NO: 26); GFP (siRNA) sense 5′-UCC CGG CUA UGU GCA GGAGdTdT-3′ (SEQ ID NO: 27) and antisense strand 5′-CUCCUG CAC AUA GCC GGGAdTdT-3′ (SEQ ID NO: 28); mMAPKAP kinase-2 (siRNA), sense 5′-CGA UGC GUGUUG ACU AUG AdTdT-3′ (SEQ ID NO: 29) and antisense strand 5′-UCA UAG UCAACA CGC AUC GdTdT-3′ (SEQ ID NO: 30); hMAPKAP kinase-2 (siRNA), sense5′-UGA CCA UCA CCG AGU UWA UdTdT-3′ (SEQ ID NO: 31) and anti-sensestrand 5′-AUA AAC UCG GUG AUG GUC AdTdT-3′ (SEQ ID NO: 32); Chk1(siRNA), 5′-UGG CAA CAG UAU UUC GGU AdTdT-3′ (SEQ ID NO: 33) andantisense strand 5′-UAC CGA AAU ACU GUU GCC AdTdT-3′ (SEQ ID NO: 34).

For overexpression studies, FLAG-6×His-tagged human Chk1 cDNA was PCRamplified and subcloned into the Mlu-1 and Not-1 sites of pHURRAdownstream from the CMV promoter. pHURRA was a kind gift from Dr. H.Pavenstadt (U. of Munster).

Therapy

Therapy according to the invention may be performed alone or inconjunction with another therapy and may be provided at home, thedoctor's office, a clinic, a hospital's outpatient department, or ahospital. Treatment generally begins at a hospital so that the doctorcan observe the therapy's effects closely and make any adjustments thatare needed. The duration of the therapy depends on the age and conditionof the patient, the stage of the patient's disease or disorder, and howthe patient responds to the treatment. Additionally, a person having agreater risk of developing a disease or disorder that may be treated bythe methods of the invention (e.g., a person who is geneticallypredisposed) may receive prophylactic treatment to inhibit or delaysymptoms of the disease. Drug administration may be performed atdifferent intervals (e.g., daily, weekly, or monthly). Therapy may begiven in on-and-off cycles that include rest periods so that thepatient's body has a chance to build healthy new cells and regain itsstrength. Therapy may be used to extend the patient's lifespan.

For cancer treatment, depending on the type of cancer and its stage ofdevelopment, the therapy can be used to slow the spreading of thecancer, to slow the cancer's growth, to kill or arrest cancer cells thatmay have spread to other parts of the body from the original tumor, torelieve symptoms caused by the cancer, or to prevent cancer in the firstplace.

Administration of Therapeutic Compounds

By selectively disrupting or preventing a compound from binding to itsnatural partner(s) through its binding site, one may inhibit thebiological activity or the biological function of a MAPKAP kinase-2polypeptide. The methods of the invention feature the use of compoundsthat inhibit an activity of a MAPKAP kinase-2 polypeptide, whether byreducing expression of the polypeptide (e.g., RNAi or antisensetherapy), or by binding directly to a MAPKAP kinase-2 polypeptide andinhibiting its substrate-binding activity. In some instances, MAPKAPkinase-2 inhibitory compounds are administered to patients having one ormore p53-deficient cells, e.g., tumor cells. Exemplary inhibitorycompounds will be described further below.

Diseases or disorders characterized by inappropriate cell cycleregulation include cellular proliferative disorders, such as neoplasias.Examples of neoplasias include, without limitation, acoustic neuroma,acute leukemia, acute lymphocytic leukemia, acute monocytic leukemia,acute myeloblastic leukemia, acute myelocytic leukemia, acutemyelomonocytic leukemia, acute promyelocytic leukemia, acuteerythroleukemia, adenocarcinoma, angiosarcoma, astrocytoma, basal cellcarcinoma, bile duct carcinoma, bladder carcinoma, brain cancer, breastcancer, bronchogenic carcinoma, cervical cancer, chondrosarcoma,chordoma, choriocarcinoma, chronic leukemia, chronic lymphocyticleukemia, chronic myelocytic leukemia, colon cancer, colon carcinoma,craniopharyngioma, cystadenocarcinoma, embryonal carcinoma,endotheliosarcoma, ependymoma, epithelial carcinoma, Ewing's tumor,glioma, heavy chain disease, hemangioblastoma, hepatoma, Hodgkin'sdisease, large cell carcinoma, leiomyosarcoma, liposarcoma, lung cancer,lung carcinoma, lymphangioendotheliosarcoma, lymphangiosarcoma,macroglobulinemia, medullary carcinoma, medulloblastoma, melanoma,meningioma, mesothelioma, myxosarcoma, neuroblastoma, non-Hodgkin'sdisease, oligodendroglioma, osteogenic sarcoma, ovarian cancer,pancreatic cancer, papillary adenocarcinomas, papillary carcinoma,pinealoma, polycythemia vera, prostate cancer, rhabdomyosarcoma, renalcell carcinoma, retinoblastoma, schwannoma, sebaceous gland carcinoma,seminoma, small cell lung carcinoma, squamous cell carcinoma, sweatgland carcinoma, synovioma, testicular cancer, uterine cancer,Waldenstrom's fibrosarcoma, and Wilm's tumor. Any of these diseases ordisorders can include, or be associated with, one or more p53-deficientcells, e.g., tumor cells.

A MAPKAP kinase-2-binding peptide, small molecule, or other compound maybe administered within a pharmaceutically-acceptable diluent, carrier,or excipient, in unit dosage form. Conventional pharmaceutical practicemay be employed to provide suitable formulations or compositions toadminister the compounds to patients suffering from a disease that iscaused by excessive cell proliferation. Administration may begin beforethe patient is symptomatic. Any appropriate route of administration maybe employed, for example, administration may be parenteral, intravenous,intra-arterial, subcutaneous, intramuscular, intracranial, intraorbital,ophthalmic, intraventricular, intracapsular, intraspinal,intracisternal, intraperitoneal, intranasal, aerosol, suppository, ororal administration. For example, therapeutic formulations may be in theform of liquid solutions or suspensions; for oral administration,formulations may be in the form of tablets or capsules; and forintranasal formulations, in the form of powders, nasal drops, oraerosols.

Combination Therapy

As described above, if desired, treatment with compounds that inhibitMAPKAP kinase-2 polypeptides may be combined with therapies for thetreatment of proliferative disease, such as radiotherapy, surgery, orchemotherapy. Chemotherapeutic agents that may be administered withcompounds (e.g., UCN-01) that interact with a MAPKAP kinase-2polypeptide include, but are not limited to, alemtuzumab, altretamine,aminoglutethimide, amsacrine, anastrozole, azacitidine, bleomycin,bicalutamide, busulfan, capecitabine, carboplatin, carmustine,celecoxib, chlorambucil, 2-chlorodeoxyadenosine, cisplatin, colchicine,cyclophosphamide, cytarabine, cytoxan, dacarbazine, dactinomycin,daunorubicin, docetaxel, doxorubicin, epirubicin, estramustinephosphate, etodolac, etoposide, exemestane, floxuridine, fludarabine,5-fluorouracil, flutamide, formestane, gemcitabine, gentuzumab,goserelin, hexamethylmelamine, hydroxyurea, hypericin, ifosfamide,imatinib, interferon, irinotecan, letrozole, leuporelin, lomustine,mechlorethamine, melphalen, mercaptopurine, 6-mercaptopurine,methotrexate, mitomycin, mitotane, mitoxantrone, nilutamide, nocodazole,paclitaxel, pentostatin, procarbazine, raltitrexed, rituximab,rofecoxib, streptozocin, tamoxifen, temozolomide, teniposide,6-thioguanine, topotecan, toremofine, trastuzumab, vinblastine,vincristine, vindesine, and vinorelbine. One or more chemotherapeuticagents may be administered in combination with one or more compoundsthat inhibit MAPKAP kinase-2 polypeptides. In some instances,combination therapy is administered to patients having one or morep53-deficient cells, e.g., tumor cells.

In the combination therapies of the invention, the therapy componentsare administered simultaneously, or within twenty-eight days of eachother, in amounts sufficient to inhibit the growth of said neoplasm.

Depending on the type of cancer and its stage of development, thecombination therapy can be used to treat cancer, to slow the spreadingof the cancer, to slow the cancer's growth, to kill or arrest cancercells that may have spread to other parts of the body from the originaltumor, to relieve symptoms caused by the cancer, or to prevent cancer inthe first place. Combination therapy can also help people live morecomfortably by eliminating cancer cells that cause pain or discomfort.

The administration of a combination of the present invention allows forthe administration of lower doses of each compound, providing similarefficacy and lower toxicity compared to administration of eithercompound alone. Alternatively, such combinations result in improvedefficacy in treating neoplasms with similar or reduced toxicity.

RNA Interference Therapy

The invention features the novel and therapeutically important discoverythat the use of RNA interference (RNAi) to reduce MAPKAP kinase-2expression renders cells more susceptible to chemotherapeutic agents.Accordingly, using the methods of the invention, nucleobase oligomersmay be employed in double-stranded RNAs for RNAi-mediated knockdown ofMAPKAP kinase-2 expression. RNAi is a method for decreasing the cellularexpression of specific proteins of interest (reviewed in Tuschl,Chembiochem 2:239-245, 2001; Sharp, Genes & Devel. 15:485-490, 2000;Hutvagner and Zamore, Curr. Opin. Genet. Devel. 12:225-232, 2002; andHannon, Nature 418:244-251, 2002). In RNAi, gene silencing is typicallytriggered post-transcriptionally by the presence of double-stranded RNA(dsRNA) in a cell. This dsRNA is processed intracellularly into shorterpieces called small interfering RNAs (siRNAs). The introduction ofsiRNAs into cells either by transfection of dsRNAs or through expressionof siRNAs using a plasmid-based expression system is increasingly beingused to create loss-of-function phenotypes in mammalian cells.

In one embodiment of the invention, a double-stranded RNA (dsRNA)molecule is made. The dsRNA can be two distinct strands of RNA that haveduplexed, or a single RNA strand that has self-duplexed (small hairpin(sh)RNA). Typically, dsRNAs are about 21 or 22 base pairs, but may beshorter or longer (up to about 29 nucleobases) if desired. dsRNA can bemade using standard techniques (e.g., chemical synthesis or in vitrotranscription). Kits are available, for example, from Ambion (Austin,Tex.) and Epicentre (Madison, Wis.). Methods for expressing dsRNA inmammalian cells are described in Brummelkamp et al. Science 296:550-553,2002; Paddison et al. Genes & Devel. 16:948-958, 2002. Paul et al.Nature Biotechnol. 20:505-508, 2002; Sui et al. Proc. Natl. Acad. Sci.USA 995515-5520, 2002; Yu et al. Proc. Natl. Acad. Sci. USA99:6047-6052, 2002; Miyagishi et al. Nature Biotechnol. 20:497-500,2002; and Lee et al. Nature Biotechnol. 20:500-505 2002, each of whichis hereby incorporated by reference. Small hairpin RNAs consist of astem-loop structure with optional 3′ UU-overhangs. While there may bevariation, stems can range from twenty-one to thirty-one base pairs(desirably twenty-five to twenty-nine base pairs), and the loops canrange from four to thirty base pairs (desirably four to twenty-threebase pairs). For expression of shRNAs within cells, plasmid vectorscontaining, e.g., the polymerase III H1-RNA or U6 promoter, a cloningsite for the stem-looped RNA insert, and a 4-5-thymidine transcriptiontermination signal can be employed. The Polymerase III promotersgenerally have well-defined initiation and stop sites and theirtranscripts lack poly(A) tails. The termination signal for thesepromoters is defined by the polythymidine tract, and the transcript istypically cleaved after the second uridine. Cleavage at this positiongenerates a 3′ UU overhang in the expressed shRNA, which is similar tothe 3′ overhangs of synthetic siRNAs. Additional methods for expressingthe shRNA in mammalian cells are described in the references citedabove.

Computer programs that employ rational design of oligos are useful inpredicting regions of the MAPKAP kinase-2 sequence that may be targetedby RNAi. For example, see Reynolds et al., Nat. Biotechnol., 22:326-330,2004, for a description of the Dharmacon siDESIGN tool. Table 3 listsseveral exemplary nucleotide sequences within MAPKAP kinase-2 that maybe targeted for purposes of RNA interference. siRNA or shRNA oligos maybe made corresponding to the sequences shown and including an overhang,e.g., a 3′ dTdT overhang and/or a loop.

TABLE 3 MAPKAP kinase-2 RNAi target sequences Sequence (5′ to 3′) SEQ IDNO: GACCAGGCATTCACAGAAA 35 TTGACCACTCCTTGTTATA 36 GACCACTCCTTGTTATACA 37TGACCATCACCGAGTTTAT 38 TCACCGAGTTTATGAACCA 39 TCAAGAAGAACGCCATCAT 40AAGCATCCGAAATCATGAA 41 AGTATCTGCATTCAATCAA 42 CTTTGACCACTCCTTGTTA 43TTTGACCACTCCTTGTTAT 44 TACGGATCGTGGATGTGTA 45 GGACGGTGGAGAACTCTTT 46CTTGTTATACACCGTACTA 47 GACGGTGGAGAACTCTTTA 48 GGAGAACTCTTTAGCCGAA 49

Antisense Therapy

As an alternative to RNAi-based approaches, therapeutic strategiesutilizing MAPKAP kinase-2 antisense nucleic acids may be employed in themethods of the invention. The technique is based on the principle thatsequence-specific suppression of gene expression can be achieved byintracellular hybridization between mRNA and a complementary antisensespecies. The formation of a hybrid RNA duplex may then interfere withthe processing/transport/translation and/or stability of the targetMAPKAP kinase-2 mRNA. Antisense strategies may use a variety ofapproaches, including the use of antisense oligonucleotides andinjection of antisense RNA. An exemplary approach features transfectionof antisense RNA expression vectors into targeted cells. Antisenseeffects can be induced by control (sense) sequences; however, the extentof phenotypic changes are highly variable. Phenotypic effects induced byantisense effects are based on changes in criteria such as proteinlevels, protein activity measurement, and target mRNA levels.

Computer programs such as OLIGO (previously distributed by NationalBiosciences Inc.) may be used to select candidate nucleobase oligomersfor antisense therapy based on the following criteria:

-   -   1) no more than 75% GC content, and no more than 75% AT content;    -   2) preferably no nucleobase oligomers with four or more        consecutive G residues (due to reported toxic effects, although        one was chosen as a toxicity control);    -   3) no nucleobase oligomers with the ability to form stable        dimers or hairpin structures; and    -   4) sequences around the translation start site are a preferred        region.        In addition, accessible regions of the target mRNA may be        predicted with the help of the RNA secondary structure folding        program MFOLD (M. Zuker, D. H. Mathews & D. H. Turner,        Algorithms and Thermodynamics for RNA Secondary Structure        Prediction: A Practical Guide. In: RNA Biochemistry and        Biotechnology, J. Barciszewski & B. F. C. Clark, eds., NATO ASI        Series, Kluwer Academic Publishers, (1999). Sub-optimal folds        with a free energy value within 5% of the predicted most stable        fold of the mRNA may be predicted using a window of 200 bases        within which a residue can find a complimentary base to form a        base pair bond. Open regions that do not form a base pair may be        summed together with each suboptimal fold, and areas that        consistently are predicted as open may be considered more        accessible to the binding to nucleobase oligomers. Additional        nucleobase oligomer that only partially fulfill some of the        above selection criteria may also be chosen as possible        candidates if they recognize a predicted open region of the        target mRNA.

Therapeutically Useful Compounds and Pharmaceutical Compositions

Any compound or pharmaceutical composition that inhibits an activity ofMAPKAP kinase-2 may be useful in the methods of the invention. The modelof the activated MAPKAP kinase-2/peptide complex described above(Table 1) indicates that peptides, or peptide-like compounds, e.g.,peptidomimetics, may be useful for inhibiting MAPKAP kinase-2. Suchcompounds achieve this effect by mimicking the natural peptide substrateof MAPKAP kinase-2 and decreasing the extent or rate with which a MAPKAPkinase-2 polypeptide is able to bind to its natural substrates in vivo.Accordingly, methods of synthesis or modification of peptides andpeptide-like compounds is described below.

Peptide Synthesis and Conjugation

A compound of the invention that includes a peptide is prepared asdetailed above. Alternatively, peptides can be prepared using standardFMOC chemistry on 2-chlorotrityl chloride resin (Int. J. Pept. Prot.Res. 38, 1991, 555-61). Cleavage from the resin is performed using 20%acetic acid in dichloromehane (DCM), which leaves the side chain stillblocked. Free terminal carboxylate peptide is then coupled to4′(aminomethy)-fluorescein (Molecular Probes, A-1351; Eugene, Oreg.)using excess diisopropylcarbodiimide (DIC) in dimethylformamide (DMF) atroom temperature. The fluorescent N—C blocked peptide is purified bysilica gel chromatography (10% methanol in DCM). The N terminal FMOCgroup is then removed using piperidine (20%) in DMF, and the N-freepeptide, purified by silica gel chromatography (20% methanol in DCM,0.5% HOAc). Finally, any t-butyl side chain protective groups areremoved using 95% trifluoroacetic acid containing 2.5% water and 2.5%triisopropyl silane. The peptide obtained in such a manner should give asingle peak by HPLC and is sufficiently pure for carrying on with theassay described below.

Peptide Modifications and Unnatural Amino Acids

It is understood that modifications can be made to the amino acidresidues of the peptide-containing compounds of the invention, toenhance or prolong the therapeutic efficacy and/or bioavailability ofthe compound. Accordingly, α-amino acids having the following generalformula (I):

where R defines the specific amino acid residue, may undergo variousmodifications. Exemplary modifications of α-amino acids, include, butare not limited to, the following formula (II):

R₁, R₂, R₃, R₄, and R₅, are independently hydrogen, hydroxy, nitro,halo, C₁₋₅ branched or linear alkyl, C₁₋₅ alkaryl, heteroaryl, and aryl;wherein the alkyl, alkaryl, heteroaryl, and aryl may be unsubstituted orsubstituted by one or more substituents selected from the groupconsisting of C₁₋₅ alkyl, hydroxy, halo, nitro, C₁₋₅ alkoxy, C₁₋₅alkylthio, trihalomethyl, C₁₋₅ acyl, arylcarbonyl, heteroarylcarbonyl,nitrile, C₁₋₅ alkoxycarbonyl, oxo, arylalkyl (wherein the alkyl grouphas from 1 to 5 carbon atoms) and heteroarylalkyl (wherein the alkylgroup has from 1 to 5 carbon atoms); alternatively, R₁ and R₂ are joinedto form a C₃₋₈ cyclic ring, optionally including oxygen, sulfur orhydrogen, or C₁₋₅ alkyl, optionally substituted by hydroxyl; or R₂ andR₃ are joined to form a C₃₋₈ cyclic ring, optionally substituted byhydroxyl and optionally including oxygen, sulfinur, C₁₋₅ aminoalkyl, orC₁₋₅ alkyl.

A compound of the invention that includes a peptide may include, but itis not limited to, an unnatural N-terminal amino acid of the formula(III):

where A¹ is an amino acid or peptide chain linked via an α-amino group;R¹ and R³ are independently hydrogen, C₁₋₅ branched or linear C₁₋₅alkyl, C₁₋₅ alkaryl, heteroaryl, and aryl, each of which areunsubstituted or substituted with a substitutent selected from: 1 to 3of C₁₋₅ alkyl, 1 to 3 of halogen, 1 to 2 of —OR⁵, N(R⁵)(R⁶), SR⁵,N—C(NR⁵)NR⁶R⁷, methylenedioxy, —S(O)_(m)R⁵, 1 to 2 of —CF₃, —OCF₃,nitro, —N(R⁵)C(O)(R⁶), —C(O)OR⁵, —C(O)N(R⁵)(R⁶), -1H-tetrazol-5-yl,—SO₂N(R⁵)(R⁶), —N(R⁵)SO₂ aryl, or —N(R⁵)SO₂R⁶; R⁵, R⁶ and R⁷ areindependently selected from hydrogen, C₁₋₅ linear or branched alkyl,C₁₋₅ alkaryl, aryl, heteroaryl, and C₃₋₇ cycloalkyl, and where two C₁₋₅alkyl groups are present on one atom, they optionally are joined to forma C₃₋₈ cyclic ring, optionally including oxygen, sulfur or NR⁷, where R⁷is hydrogen, or C₁₋₅ alkyl, optionally substituted by hydroxyl; R² ishydrogen, F, C₁₋₅ linear or branched alkyl, C₁₋₅ alkaryl; or R² and R¹are joined to form a C₃₋₈ cyclic ring, optionally including oxygen,sulfur, or NR⁷, where R⁷ is hydrogen, or C₁₋₅ alkyl, optionallysubstituted by hydroxyl, or R² and R³ are joined to form a C₃₋₈ cyclicring, optionally substituted by hydroxyl and optionally includingoxygen, sulfur or NR⁷, where R⁷ is hydrogen, or C₁₋₅ alkyl; R² ishydrogen, F, C₁₋₅ linear or branched alkyl, C₁₋₅ alkaryl; and R⁴ ishydrogen, C₁₋₅ branched or linear C₁₋₅ alkyl, C₁₋₅ alkaryl, heteroaryl,and aryl, each of which are unsubstituted or substituted with asubstitutent selected from: 1 to 3 of C₁₋₅ alkyl, 1 to 3 of halogen, 1to 2 of —OR⁵, N(R⁵)(R⁶), N—C(NR⁵)NR⁶R⁷, methylenedioxy, —S(O)_(m)R⁵(where m is 0-2), 1 to 2 of —CF₃, —OCF₃, nitro, —N(R⁵)C(O)(R⁶),—N(R⁵)C(O)(OR⁶), —C(O)OR⁵, —C(O)N(R⁵)(R⁶), -1H-tetrazol-5-yl,—SO₂N(R⁵)(R⁶), —N(R⁵)SO₂ aryl, or —N(R⁵)SO₂R⁶, R⁵, R⁶ and R⁷ areindependently selected from hydrogen, C₁₋₅ linear or branched alkyl,C₁₋₅ alkaryl, aryl, heteroaryl, and C₃₋₇ cycloalkyl, and where two C₁₋₅alkyl groups are present on one atom, they optionally are joined to forma C₃₋₈ cyclic ring, optionally including oxygen, sulfur or NR⁷, where R⁷is hydrogen, or C₁₋₅ alkyl, optionally substituted by hydroxyl.

A compound of the invention may also include an unnatural internal aminoacid of the formula:

where A² is an amino acid or peptide chain linked via an α-carboxygroup; A¹ is an amino acid or peptide chain linked via an α-amino group;R¹ and R³ are independently hydrogen, C₁₋₅ branched or linear C₁₋₅alkyl, C₁₋₅ alkaryl, heteroaryl, and aryl, each of which areunsubstituted or substituted with a substitutent selected from: 1 to 3of C₁₋₅ alkyl, 1 to 3 of halogen, 1 to 2 of —OR⁵, N(R⁵)(R⁶), SR⁵,N—C(NR⁵)NR⁶R⁷, methylenedioxy, —S(O)_(m)R⁵ (m is 1-2), 1 to 2 of —CF₃,—OCF₃, nitro, —N(R⁵)C(O)(R⁶), —C(O)OR⁵, —C(O)N(R⁵)(R⁶),-1H-tetrazol-5-yl, —SO₂N(R⁵)(R⁶), —N(R⁵)SO₂ aryl, or —N(R⁵)SO₂R⁶; R¹, R⁶and R⁷ are independently selected from hydrogen, C₁₋₅ linear or branchedalkyl, C₁₋₅ alkaryl, aryl, heteroaryl, and C₃₋₇ cycloalkyl, and wheretwo C₁₋₅ alkyl groups are present on one atom, they optionally arejoined to form a C₃₋₈ cyclic ring, optionally including oxygen, sulfuror NR⁷, where R⁷ is hydrogen, or C₁₋₅ alkyl, optionally substituted byhydroxyl; and R² is hydrogen, F, C₁₋₅ linear or branched alkyl, C₁₋₅alkaryl; or R² and R¹ are joined to form a C₃₋₈ cyclic ring, optionallyincluding oxygen, sulfur or NR⁷, where R⁷ is hydrogen, or C₁₋₅ alkyl,optionally substituted by hydroxyl, or R² and R³ are joined to form aC₃₋₈ cyclic ring, optionally substituted by hydroxyl and optionallyincluding oxygen, sulfur or NR⁷, where R⁷ is hydrogen, or C₁₋₅ alkyl.

The invention also includes modifications of the peptide-containingcompounds of the invention, wherein an unnatural internal amino acid ofthe formula:

is present, where A² is an amino acid or peptide chain linked via anα-carboxy group; A¹ is an amino acid or peptide chain linked via anα-amino group; R¹ and R³ are independently hydrogen, C₁₋₅ branched orlinear C₁₋₅ alkyl, and C₁₋₅ alkaryl; R² is hydrogen, F, C₁₋₅ linear orbranched alkyl, C₁₋₅ alkaryl; or R² and R¹ are joined to form a C₃₋₈cyclic ring, optionally including oxygen, sulfur or NR⁷, where R⁷ ishydrogen, or C₁₋₅ alkyl, optionally substituted by hydroxyl; X is O orS; and R⁵ and R⁶ are independently selected from hydrogen, C₁₋₅ linearor branched alkyl, C₁₋₅ alkaryl, aryl, heteroaryl, and C₃₋₇ cycloalkyl,and where two C₁₋₅ alkyl groups are present on one atom, they optionallyare joined to form a C₃₋₈ cyclic ring, optionally including oxygen,sulfur or NR⁷, where R⁷ is hydrogen, or C₁₋₅ alkyl, optionallysubstituted by hydroxyl; or R⁵ and R⁶ are joined to form a C₃₋₈ cyclicring, optionally including oxygen, sulfur or NR⁷, where R⁷ is hydrogen,or C₁₋₅ alkyl, optionally substituted by hydroxyl.

A compound of the invention may also include a C-terminal unnaturalinternal amino acid of the formula:

where A² is an amino acid or peptide chain linked via an α-carboxygroup; R¹ and R³ are independently hydrogen, C₁₋₅ branched or linearC₁₋₅ alkyl, C₁₋₅ alkaryl, heteroaryl, and aryl, each of which areunsubstituted or substituted with a substitutent selected from: 1 to 3of C₁₋₅ alkyl, 1 to 3 of halogen, 1 to 2 of —OR⁵, N(R⁵)(R⁶), SR⁵,N—C(NR⁵)NR⁶R⁷, methylenedioxy, S(O)_(m)R⁵, 1 to 2 of —CF₃, —OCF₃, nitro,—N(R⁵)C(O)(R⁶), —C(O)OR⁵, —C(O)N(R⁵)(R⁶), -1H-tetrazol-5-yl,—SO₂N(R⁵)(R⁶), —N(R⁵)SO₂ aryl, or —N(R⁵)SO₂R⁶; R⁵, R⁶ and R⁷ areindependently selected from hydrogen, C₁₋₅ linear or branched alkyl,C₁₋₅ alkaryl, aryl, heteroaryl, and C₃₋₇ cycloalkyl, and where two C₁₋₅alkyl groups are present on one atom, they optionally are joined to forma C₃₋₈ cyclic ring, optionally including oxygen, sulfur or NR⁷, where R⁷is hydrogen, or C₁₋₅ alkyl, optionally substituted by hydroxyl; R² ishydrogen, F, C₁₋₅ linear or branched alkyl, C₁₋₅ alkaryl; or R² and R¹are joined to form a C₃₋₈ cyclic ring, optionally including oxygen,sulfur or NR⁷, where R⁷ is hydrogen, or C₁₋₅ alkyl, optionallysubstituted by hydroxyl; or R² and R³ are joined to form a C₃₋₈ cyclicring, optionally substituted by hydroxyl and optionally includingoxygen, sulfur or NR⁷, where R⁷ is hydrogen, or C₁₋₅ alkyl; R² ishydrogen, F, C₁₋₅ linear or branched alkyl, C₁₋₅ alkaryl; and Q is OH,OR⁵, or NR⁵R⁶, where R⁵, R⁶ are independently selected from hydrogen,C₁₋₅ linear or branched alkyl, C₁₋₅ alkaryl, aryl, heteroaryl, and C₃₋₇cycloalkyl, and where two C₁₋₅ alkyl groups are present on one atom,they optionally are joined to form a C₃₋₈ cyclic ring, optionallyincluding oxygen, sulfur or NR⁷, where R⁷ is hydrogen, or C₁₋₅ alkyl,optionally substituted by hydroxyl. Methods well known in the art formodifying peptides are found, for example, in “Remington: The Scienceand Practice of Pharmacy,” (20th ed., ed. A. R. Gennaro, 2000,Lippincott Williams & Wilkins, Philadelphia).

Peptidomimetics

Peptide derivatives (e.g., peptidomimetics) include cyclic peptides,peptides obtained by substitution of a natural amino acid residue by thecorresponding D-stereoisomer, or by an unnatural amino acid residue,chemical derivatives of the peptides, dual peptides, multimers of thepeptides, and peptides fused to other proteins or carriers. A cyclicderivative of a peptide of the invention is one having two or moreadditional amino acid residues suitable for cyclization. These residuesare often added at the carboxyl terminus and at the amino terminus. Apeptide derivative may have one or more amino acid residues replaced bythe corresponding D-amino acid residue. In one example, a peptide orpeptide derivative of the invention is all-L, all-D, or a mixedD,L-peptide. In another example, an amino acid residue is replaced by anunnatural amino acid residue. Examples of unnatural or derivatizedunnatural amino acids include Na-methyl amino acids, Cα-methyl aminoacids, and β-methyl amino acids.

A chemical derivative of a peptide of the invention includes, but is notlimited to, a derivative containing additional chemical moieties notnormally a part of the peptide. Examples of such derivatives include:(a) N-acyl derivatives of the amino terminal or of another free aminogroup, where the acyl group may be either an alkanoyl group, e.g.,acetyl, hexanoyl, octanoyl, an aroyl group, e.g., benzoyl, or a blockinggroup such as Fmoc (fluorenylmethyl-O—CO—), carbobenzoxy(benzyl-O—CO—),monomethoxysuccinyl, naphthyl-NH—CO—, acetylamino-caproyl,adamantyl-NH—CO—; (b) esters of the carboxyl terminal or of another freecarboxyl or hydroxy groups; (c) amides of the carboxyl terminal or ofanother free carboxyl groups produced by reaction with ammonia or with asuitable amine; (d) glycosylated derivatives; (e) phosphorylatedderivatives; (f) derivatives conjugated to lipophilic moieties, e.g.,caproyl, lauryl, stearoyl; and (g) derivatives conjugated to an antibodyor other biological ligand. Also included among the chemical derivativesare those derivatives obtained by modification of the peptide bond—CO—NH—, for example, by: (a) reduction to —CH₂—NH—; (b) alkylation to—CO—N(alkyl)—; and (c) inversion to —NH—CO—. Peptidomimetics may alsocomprise phosphonate or sulfonate moieties.

A dual peptide of the invention consists of two of the same, or twodifferent, peptides of the invention covalently linked to one another,either directly or through a spacer.

Multimers of the invention consist of polymer molecules formed from anumber of the same or different peptides or derivatives thereof.

In one example, a peptide derivative is more resistant to proteolyticdegradation than the corresponding non-derivatized peptide. For example,a peptide derivative having D-amino acid substitution(s) in place of oneor more L-amino acid residue(s) resists proteolytic cleavage.

In another example, the peptide derivative has increased permeabilityacross a cell membrane as compared to the corresponding non-derivatizedpeptide. For example, a peptide derivative may have a lipophilic moietycoupled at the amino terminus and/or carboxyl terminus and/or aninternal site. Such derivatives are highly preferred when targetingintracellular protein-protein interactions, provided they retain thedesired functional activity.

In another example, a peptide derivative binds with increased affinityto a ligand (e.g., a MAPKAP kinase-2 polypeptide).

The peptides or peptide derivatives of the invention are obtained by anymethod of peptide synthesis known to those skilled in the art, includingsynthetic and recombinant techniques. For example, the peptides orpeptide derivatives can be obtained by solid phase peptide synthesiswhich, in brief, consists of coupling the carboxyl group of theC-terminal amino acid to a resin and successively adding N-alphaprotected amino acids. The protecting groups may be any such groupsknown in the art. Before each new amino acid is added to the growingchain, the protecting group of the previous amino acid added to thechain is removed. The coupling of amino acids to appropriate resins hasbeen described by Rivier et al. (U.S. Pat. No. 4,244,946). Such solidphase syntheses have been described, for example, by Merrifield, J. Am.Chem. Soc. 85:2149, 1964; Vale et al., Science 213:1394-1397, 1984;Marki et al., J. Am. Chem. Soc. 10:3178, 1981, and in U.S. Pat. Nos.4,305,872 and 4,316,891. Desirably, an automated peptide synthesizer isemployed.

Purification of the synthesized peptides or peptide derivatives iscarried out by standard methods, including chromatography (e.g., ionexchange, affinity, and sizing column chromatography), centrifugation,differential solubility, hydrophobicity, or by any other standardtechnique for the purification of proteins. In one embodiment, thinlayer chromatography is employed. In another embodiment, reverse phaseHPLC (high performance liquid chromatography) is employed.

Finally, structure-function relationships determined from the peptides,peptide derivatives, and other small molecules of the invention may alsobe used to prepare analogous molecular structures having similarproperties. Thus, the invention is contemplated to include molecules inaddition to those expressly disclosed that share the structure,hydrophobicity, charge characteristics and side chain properties of thespecific embodiments exemplified herein.

In one example, such derivatives or analogs that have the desiredbinding activity can be used for binding to a molecule or other targetof interest, such as any MAPKAP kinase-2 polypeptide. Derivatives oranalogs that retain, or alternatively lack or inhibit, a desiredproperty-of-interest (e.g., inhibit MAPKAP kinase-2 binding to a naturalligand), can be used to inhibit the biological activity of a MAPKAPkinase-2 polypeptide.

In particular, peptide derivatives are made by altering amino acidsequences by substitutions, additions, or deletions that provide forfunctionally equivalent molecules, or for functionally enhanced ordiminished molecules, as desired. Due to the degeneracy of the geneticcode, other nucleic acid sequences that encode substantially the sameamino acid sequence may be used for the production of recombinantpeptides. These include, but are not limited to, nucleotide sequencescomprising all or portions of a peptide of the invention that is alteredby the substitution of different codons that encode a functionallyequivalent amino acid residue within the sequence, thus producing asilent change.

The derivatives and analogs of the invention can be produced by variousmethods known in the art. The manipulations that result in theirproduction can occur at the gene or protein level. For example, a clonednucleic acid sequence can be modified by any of numerous strategiesknown in the art (Sambrook et al., 1989, Molecular Cloning, A LaboratoryManual, 2d ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor,N.Y.). The sequence can be cleaved at appropriate sites with restrictionendonuclease(s), followed by further enzymatic modification if desired,isolated, and ligated in vitro.

MAPKAP Kinase-2 Inhibitors

Based on the present discovery that RNAi knockdown of MAPKAP kinase-2expression sensitizes cells to chemotherapeutic agents, any compoundthat inhibits MAPKAP kinase-2, whether specifically or nonspecifically,may be of utility in antineoplastic therapy. Suitable MAPKAP kinase-2inhibitors that may be used in the methods and compositions of theinvention include UCN-01,2-(3-aminopropyl)-8-(methylthio)-2,4,5,6-tetrahydropyrazolo[3,4-e]indazole-3-carboxylicacid dihydrochloride,2-(3-aminopropyl)-2,4,5,6-tetrahydropyrazolo[3,4-e]indazole-3-carboxylicacid hydrochloride,2-(3-{[2-(4-bromophenyl)ethyl]amino}propyl)-2,4,5,6-tetrahydropyrazolo[3,4-e]indazole-3-carboxylicacid hydrochloride,2-(2-aminoethyl)-2,4,5,6-tetrahydropyrazolo[3,4-e]indazole-3-carboxylicacid hydrochloride,8-(allylthio)-2-(3-aminopropyl)-2,4,5,6-tetrahydropyrazolo[3,4-e]indazole-3-carboxylicacid dihydrochloride,2-(3-aminopropyl)-8-(benzylthio)-2,4,5,6-tetrahydropyrazolo[3,4-e]indazole-3-carboxylicacid dihydrochloride,2-{3-[(2-thien-2-ylethyl)amino]propyl}-2,4,5,6-tetrahydropyrazolo[3,4-e]indazole-3-carboxylicacid,2-{3-[(2-thien-3-ylethyl)amino]propyl}-2,4,5,6-tetrahydropyrazolo[3,4-e]indazole-3-carboxylicacid hydrochloride, ethyl2-(3-{[2-(4-bromophenyl)ethyl]amino}propyl)-2,4,5,6-tetrahydropyrazolo[3,4-e]indazole-3-carboxylate,2-(3-aminopropyl)-8-quinolin-3-yl-4,5-dihydro-2H-pyrazolo[4,3-h]quinazoline-3-carboxylicacid dihydrochloride,2-(3-aminopropyl)-8-(1,3-benzodioxol-5-yl)-4,5-dihydro-2H-pyrazolo[4,3-h]quinazoline-3-carboxylicacid hydrochloride,2-(3-aminopropyl)-8-phenyl-4,5-dihydro-2H-pyrazolo[4,3-h]quinazoline-3-carboxylicacid hydrochloride,2-quinolin-3-yl-5,6,8,9,10,11-hexahydro-7H-[1,4]diazepino[1′,2′:1,5]pyrazolo[4,3-h]quinazolin-7-one,2-pyridin-3-yl-5,6,8,9,10,11-hexahydro-7H-(1,4]diazepino[1′,2′:1,5]pyrazolo[4,3-h]quinazolin-7-one,8-quinolin-3-yl-2-[3-(tritylamino)propyl]-4,5-dihydro-2H-pyrazolo[4,3-h]quinazoline-3-carboxylicacid hydrochloride,2-(1,3-benzodioxol-5-yl)-5,6,8,9,10,11-hexahydro-7H-[1,4]diazepino[1′,2′:1,5]pyrazolo[4,3-h]quinazolin-7-one,2-(4-methoxyphenyl)-5,6,8,9,10,11-hexahydro-7H-[1,4]diazepino[1′,2′:1,5]pyrazolo[4,3-h]quinazolin-7-one,2-pyridin-4-yl-5,6,8,9,10,11-hexahydro-7H-[1,4]diazepino[1′,2′:1,5]pyrazolo[4,3-h]quinazolin-7-onehydrochloride,2-(3-aminopropyl)-8-quinolin-3-yl-4,5-dihydro-2H-pyrazolo[3,4-f]isoquinoline-3-carboxylicacid trifluoroacetate,2-(3-aminopropyl)-8-(3-nitrophenyl)-4,5-dihydro-2H-pyrazolo[3,4-f]isoquinoline-3-carboxylicacid trifluoroacetate,2-(3-aminopropyl)-8-(4-hydroxyphenyl)-4,5-dihydro-2H-pyrazolo[3,4-f]isoquinoline-3-carboxylicacid hydrobromide,2-(3-aminopropyl)-8-(3-hydroxyphenyl)-4,5-dihydro-2H-pyrazolo[3,4-f]isoquinoline-3-carboxylicacid hydrobromide,2-(3-aminopropyl)-8-(2-naphthyl)-4,5-dihydro-2H-pyrazolo[3,4-q]isoquinoline-3-carboxylicacid trifluoroacetate,2-(3-aminopropyl)-8-(3,5-difluorophenyl)-4,5-dihydro-2H-pyrazolo[3,4-f]isoquinoline-3-carboxylicacid trifluoroacetate,2-(3-aminopropyl)-8-(1,3-benzodioxol-5-yl)-4,5-dihydro-2H-pyrazolo[3,4-q]isoquinoline-3-carboxylicacid trifluoroacetate,2-(3-aminopropyl)-8-(3-cyanophenyl)-4,5-dihydro-2H-pyrazolo[3,4-q]isoquinoline-3-carboxylicacid trifluoroacetate,9-(hydroxymethyl)-2-quinolin-3-yl-5,6,9,10-tetrahydropyrazino[1′,2′:1,5]pyrazolo[3,4-f]isoquinolin-7(8H)-onetrifluoroacetate,2-(3-aminopropyl)-8-(4-methoxyphenyl)-4,5-dihydro-2H-pyrazolo[3,4-q]isoquinoline-3-carboxylicacid trifluoroacetate,2-(3-aminopropyl)-8-[3-(methyl-sulfonyl)phenyl]-4,5-dihydro-2H-pyrazolo[3,4-q]isoquinoline-3-carboxylicacid trifluoroacetate,2-(3-aminopropyl)-8-[3-(trifluoromethyl)phenyl]-4,5-dihydro-2H-pyrazolo[3,4-q]isoquinoline-3-carboxylicacid trifluoroacetate,2-(1H-imidazol-1-yl)-5,6,9,10-tetrahydropyrazino[1′,2′:1,5]pyrazolo[3,4-q]isoquinolin-7(8H)-onetrifluoroacetate,2-(3-aminopropyl)-8-(3-methoxyphenyl)-4,5-dihydro-2H-pyrazolo[3,4-f]isoquinoline-3-carboxylicacid trifluoroacetate,2-(3-aminopropyl)-8-[4-(trifluoromethyl)phenyl]-4,5-dihydro-2H-pyrazolo[3,4-f]isoquinoline-3-carboxylicacid trifluoroacetate,2-(3-aminopropyl)-8-anilino-4,5-dihydro-2H-pyrazolo[3,4-f]isoquinoline-3-carboxylicacid,2-(3-aminopropyl)-8-(3,4-difluorophenyl)-4,5-dihydro-2H-pyrazolo[3,4-f]isoquinoline-3-carboxylicacid trifluoroacetate,2-(3-{[2-(4′-carboxy-1,1′-biphenyl-4-yl)ethyl]amino}propyl)-4,5-dihydro-2H-pyrazolo[3,4-f]isoquinoline-3-carboxylicacid trifluoroacetate,2-(4-hydroxyphenyl)-5,6,9,10-tetrahydropyrazino[1′,2′:1,5]pyrazolo[3,4-f]isoquinolin-7(8H)-onetrifluoroacetate,2-propyl-8-quinolin-3-yl-4,5-dihydro-2H-pyrazolo[3,4-f]isoquinoline-3-carboxylicacid,2-[3-({2-[3′-(trifluoromethyl)-1,1′-biphenyl-4-yl]ethyl}amino)propyl]-4,5-dihydro-2H-pyrazolo[3,4-f]isoquinoline-3-carboxylicacid trifluoroacetate,2-(3-aminopropyl)-8-(4-tert-butylphenyl)-4,5-dihydro-2H-pyrazolo[3,4-f]isoquinoline-3-carboxylicacid trifluoroacetate,2-[3-({2-[4-(3-furyl)phenyl]ethyl}amino)propyl]-4,5-dihydro-2H-pyrazolo[3,4-f]isoquinoline-3-carboxylicacid trifluoroacetate,2-(3-{[2-(3′-chloro-1,1′-biphenyl-4-yl)ethyl]amino}propyl)-4,5-dihydro-2H-pyrazolo[3,4-f]isoquinoline-3-carboxylicacid trifluoroacetate,2-(4-methoxyphenyl)-5,6,8,9,10,1′-hexahydro-7H-[1,4]diazepino[1′,2′:1,5]pyrazolo[3,4-f]isoquinolin-7-one,2-[3-({2-[4′-(trifluoromethyl)-1,1′-biphenyl-4-yl]ethyl}amino)propyl]-4,5-dihydro-2H-pyrazolo[3,4-f]isoquinoline-3-carboxylicacid trifluoroacetate,2-(3-hydroxyphenyl)-5,6,9,10-tetrahydropyrazino[1′,2′-:1,5]pyrazolo[3,4-f]isoquinolin-7(8H)-one,2-(3-aminopropyl)-N-hydroxy-8-quinolin-3-yl-4,5-dihydro-2H-pyrazolo[3,4-f-]isoquinoline-3-carboxamidehydrochloride,2-[(E)-2-(4-hydroxyphenyl)ethenyl]-5,6,9,10-tetrahydropyrazino[1′,2′:1,5]pyrazolo[3,4-f]isoquinolin-7(8H)-onetrifluoroacetate,2-quinolin-3-yl-5,6,8,9,10,1′-hexahydro-7H-[1,4]diazepino[1′,2′:1,5]pyrazolo[3,4-f]isoquinolin-7-one,2-(4-hydroxyphenyl)-5,6,8,9,10,11-hexahydro-7H-[1,4]diazepino[1′,2′:1,5]pyrazolo[3,4-f]isoquinolin-7-one,2-(3-{[2-(2′-chloro-1,1′-biphenyl-4-yl)ethyl]amino}propyl)-4,5-dihydro-2H-pyrazolo[3,4-f]isoquinoline-3-carboxylicacid trifluoroacetate,2-(3-{[2-(4′-tert-butyl-1,1′-biphenyl-4-yl)ethyl]amino}propyl)-4,5-dihydro-2H-pyrazolo[3,4-f]isoquinoline-3-carboxylicacid trifluoroacetate,2-(3-{[2-(3,4-dichlorophenyl)ethyl]amino}propyl)-4,5-dihydro-2H-pyrazolo[3,4-f]isoquinoline-3-carboxylicacid trifluoroacetate,2-(3-{[3-(3-chlorophenyl)propyl]amino}propyl)-4,5-dihydro-2H-pyrazolo[3,4-f]isoquinoline-3-carboxylicacid trifluoroacetate,2-[(E)-2-phenylethenyl]-5,6,9,10-tetrahydropyrazino[1′,2′:1,5]pyrazolo[3,4-f]isoquinolin-7(8H)-onetrifluoroacetate,2-(3-{[2-(4-pyridin-4-ylphenyl)ethyl]amino}propyl)-4,5-dihydro-2H-pyrazolo[3,4-f]isoquinoline-3-carboxylicacid trifluoroacetate,2-(3-{[3-(4-bromophenyl)propyl]amino}propyl)-4,5-dihydro-2H-pyrazolo[3,4-f]isoquinoline-3-carboxylicacid trifluoroacetate,2-(3-{[3-(4-tert-butylphenyl)propyl]amino}propyl)-4,5-dihydro-2H-pyrazolo[3,4-f]isoquinoline-3-carboxylicacid trifluoroacetate,2-(3-{[2-(3′-isopropyl-1,1′-biphenyl-4-yl)ethyl]amino}propyl)-4,5-dihydro-2H-pyrazolo[3,4-f]isoquinoline-3-carboxylicacid trifluoroacetate,2-{3-[(2-thien-2-ylethyl)amino]propyl}-4,5-dihydro-2H-pyrazolo[3,4-f]isoquinoline-3-carboxylicacid trifluoroacetate,2-[4-(dimethylamino)phenyl]-5,6,9,10-tetrahydropyrazino[1′,2′:1,5]pyrazolo[3,4-f]isoquinolin-7(8H)-one,2-(3-{[2-(1,1′-biphenyl-4-yl)ethyl]amino}propyl)-4,5-dihydro-2H-pyrazolo[3,4-f]isoquinoline-3-carboxylicacid trifluoroacetate,2-(3-methoxyphenyl)-5,6,9,10-tetrahydropyrazino[1′,2′:1,5]pyrazolo[3,4-f]-isoquinolin-7(8H)-one,2-(3-{[2-(4-bromophenyl)ethyl]amino}propyl)-4,5-dihydro-2H-pyrazolo[3,4-f]isoquinoline-3-carboxylicacid trifluoroacetate,2-(3-{[2-(2,4-dichlorophenyl)ethyl]amino}propyl)-4,5-dihydro-2H-pyrazolo[3,4-f]isoquinoline-3-carboxylicacid trifluoroacetate,2-{3-[(benzylsulfonyl)amino]propyl}-8-quinolin-3-yl-4,5-dihydro-2H-pyrazolo[3,4-f]isoquinoline-3-carboxylicacid,9-(aminomethyl)-5,6,9,10-tetrahydropyrazino[1′,2′:1,5]pyrazolo[3,4-f]isoquinolin-7(8H)-onetrifluoroacetate,2-(3-nitrophenyl)-5,6,8,9,10,1′-hexahydro-7H-[1,4]diazepino[1′,2′:1,5]pyrazolo[3,4-f]isoquinolin-7-one,2-(3-aminopropyl)-8-quinolin-3-yl-4,5-dihydro-2H-pyrazolo[3,4-f]isoquinoline-3-carboxamidehydrochloride,2-(3-{[3-(4-chlorophenyl)propyl]amino}propyl)-4,5-dihydro-2H-pyrazolo[3,4-f]isoquinoline-3-carboxylicacid trifluoroacetate,2-[3-(dimethylamino)phenyl]-5,6,8,9,10,11-hexahydro-7H-[1,4]diazepino[1′,2′:1,5]pyrazolo[3,4-f]isoquinolin-7-one,2-(3-{[(4-chlorobenzyl)sulfonyl]amino}propyl)-8-quinolin-3-yl-4,5-dihydro-2H-pyrazolo[3,4-f]isoquinoline-3-carboxylicacid,2-(3-{[2-(4-pyridin-3-ylphenyl)ethyl]amino}propyl)-4,5-dihydro-2H-pyrazolo[3,4-f]isoquinoline-3-carboxylicacid trifluoroacetate,2-(3-{[2-(4-chlorophenyl)ethyl]amino}propyl)-4,5-dihydro-2H-pyrazolo[3,4-f]isoquinoline-3-carboxylicacid trifluoroacetate,2-(3-{[2-(5-chlorothien-2-yl)ethyl]amino}propyl)-4,5-dihydro-2H-pyrazolo[3,4-f]isoquinoline-3-carboxylicacid trifluoroacetate,2-(3-{[3-(4-cyanophenyl)propyl]amino}propyl)-4,5-dihydro-2H-pyrazolo[3,4-f]isoquinoline-3-carboxylicacid trifluoroacetate,2-(3-{[3-(5-methyl-2-furyl)butyl]amino}propyl)-4,5-dihydro-2H-pyrazolo[3,4-f]isoquinoline-3-carboxylicacid trifluoroacetate,2-[3-({2-[4-(1-benzothien-3-yl)phenyl]ethyl}amino)propyl]-4,5-dihydro-2H-pyrazolo[3,4-f]isoquinoline-3-carboxylicacid trifluoroacetate,2-(3-ammoniopropyl)-3-carboxy-4,5-dihydro-2H-pyrazolo[3,4-f]isoquinolin-7-iumdichloride,2-(4-methoxyphenyl)-5,6,9,10-tetrahydropyrazino[1′,2′:1,5]pyrazolo[3,4-f]isoquinolin-7(8H)-one,2-(3-{[3-(4-acetylphenyl)propyl]amino}propyl)-4,5-dihydro-2H-pyrazolo[3,4-f]isoquinoline-3-carboxylicacid trifluoroacetate,2-(3-aminopropyl)-4,5-dihydro-2H-pyrazolo[3,4-f]isoquinoline-3-carboxylicacid trifluoroacetate,2-[3-({3-[4-(methylsulfonyl)phenyl]propyl}amino)propyl]-4,5-dihydro-2H-pyrazolo[3,4-f]isoquinoline-3-carboxylicacid trifluoroacetate,2-(3-aminopropyl)-8-(2-methoxyphenyl)-4,5-dihydro-2H-pyrazolo[3,4-f]isoquinoline-3-carboxylicacid trifluoroacetate,2-[3-({2-[3′-(aminomethyl)-1,1′-biphenyl-4-yl]ethyl}amino)propyl]-4,5-dihydro-2H-pyrazolo[3,4-f]isoquinoline-3-carboxylicacid trifluoroacetate,2-(2-aminoethyl)-4,5-dihydro-2H-pyrazolo[3,4-f]isoquinoline-3-carboxylicacid dihydrochloride,2-(3-{[2-(4-nitrophenyl)ethyl]amino}propyl)-4,5-dihydro-2H-pyrazolo[3,4-f]isoquinoline-3-carboxylicacid trifluoroacetate,2-[3-({2-[2′-(trifluoromethyl)-1,1′-biphenyl-4-yl]ethyl}amino)propyl]-4,5-dihydro-2H-pyrazolo[3,4-f]isoquinoline-3-carboxylicacid trifluoroacetate,9-(hydroxymethyl)-5,6,9,10-tetrahydropyrazino[1′,2′:1,5]pyrazolo[3,4-f]isoquinolin-7(8H)-one,2-(3-{[2-(4-methylphenyl)ethyl]amino}propyl)-4,5-dihydro-2H-pyrazolo[3,4-f]isoquinoline-3-carboxylicacid trifluoroacetate,9-{[(2-thien-2-ylethyl)amino]methyl}-5,6,9,10-tetrahydropyrazino[1′,2′:1,5]pyrazolo[3,4-f]isoquinolin-7(8H)-one,2-(3-{[2-(4-ethoxyphenyl)ethyl]amino}propyl)-4,5-dihydro-2H-pyrazolo[3,4-f]isoquinoline-3-carboxylicacid trifluoroacetate,2-(1,3-benzodioxol-5-yl)-5,6,8,9,10,11-hexahydro-7H-[1,4]diazepino[1′,2′:1,5]pyrazolo[3,4-f]isoquinolin-7-one,2-(3-{[2-(4-methoxyphenyl)ethyl]amino}propyl)-4,5-dihydro-2H-pyrazolo[3,4-f]isoquinoline-3-carboxylicacid trifluoroacetate,3-[3-(1H-tetraazol-5-yl)-4,5-dihydro-2H-pyrazolo[3,4-f]isoquinolin-2-yl]propan-1-aminehydrochloride,2-(3-aminopropyl)-8-chloro-4,5-dihydro-2H-pyrazolo[3,4-q]isoquinoline-3-carboxylicacid trifluoroacetate,2-(3-{[(1R,2S)-2-phenylcyclopropyl]amino}propyl)-4,5-dihydro-2H-pyrazolo[3,4-f]isoquinoline-3-carboxylicacid trifluoroacetate,2-{3-[(3,3-diphenylpropyl)amino]propyl}-4,5-dihydro-2H-pyrazolo[3,4-f]isoquinoline-3-carboxylicacid trifluoroacetate,2-(3-{[2-(3-bromo-4-methoxyphenyl)ethyl]amino}propyl)-4,5-dihydro-2H-pyrazolo[3,4-f]isoquinoline-3-carboxylicacid trifluoroacetate,2-{3-[(4-phenylbutyl)amino]propyl}-4,5-dihydro-2H-pyrazolo[3,4-f]isoquinoline-3-carboxylicacid trifluoroacetate,2-[3-(2,3-dihydro-1H-inden-2-ylamino)propyl]-4,5-dihydro-2H-pyrazolo[3,4-f]isoquinoline-3-carboxylicacid trifluoroacetate,2-(2-naphthyl)-5,6,8,9,-10,11-hexahydro-7H-[1,4]diazepino[1′,2′:1,5]pyrazolo[3,4-f]isoquinolin-7-one,2-{3-[(3-phenylpropyl)amino]propyl}-4,5-dihydro-2H-pyrazolo[3,4-f]isoquinoline-3-carboxylicacid trifluoroacetate,2-(3-{[2-(4-fluorophenyl)ethyl]amino}propyl)-4,5-dihydro-2H-pyrazolo[3,4-f]isoquinoline-3-carboxylicacid trifluoroacetate,2-{3-[(2-thien-3-ylethyl)amino]propyl}-4,5-dihydro-2H-pyrazolo[3,4-f]isoquinoline-3-carboxylicacid dihydrochloride,5,6,9,10-tetrahydropyrazino[1′,2′:1,5]pyrazolo[3,4-f]isoquinolin-7(8H)-one,2-[3-(glycoloylamino)propyl]-8-quinolin-3-yl-4,5-dihydro-2H-pyrazolo[3,4-f]isoquinoline-3-carboxylicacid hydrochloride,8-quinolin-3-yl-4,5-dihydro-2H-pyrazolo[3,4-f]isoquinoline-3-carboxylicacid,2-(3-{[2-(4-ethylphenyl)ethyl]amino}propyl)-4,5-dihydro-2H-pyrazolo[3,4-f]isoquinoline-3-carboxylicacid trifluoroacetate,2-(3-{[2-(2-chlorophenyl)ethyl]amino}propyl)-4,5-dihydro-2H-pyrazolo[3,4-f]isoquinoline-3-carboxylicacid trifluoroacetate,2-{3-[(2-ethylbutyl)amino]propyl}-4,5-dihydro-2H-pyrazolo[3,4-f]isoquinoline-3-carboxylicacid trifluoroacetate,8-quinolin-3-yl-4,5-dihydro-2H-pyrazolo[3,4-f]isoquinoline-3-carboxamide,2-{3-[(2-pyridin-4-ylethyl)amino]propyl}-4,5-dihydro-2H-pyrazolo[3,4-f]isoquinoline-3-carboxylicacid trifluoroacetate,2-(3-{[2-(4-chlorophenyl)propyl]amino}propyl)-4,5-dihydro-2H-pyrazolo[3,4-f]isoquinoline-3-carboxylicacid trifluoroacetate,2-(3-{[2-(3-chlorophenyl)ethyl]amino}propyl)-4,5-dihydro-2H-pyrazolo[3,4-f]isoquinoline-3-carboxylicacid trifluoroacetate,2-[3-(glycoloylamino)propyl]-4,5-dihydro-2H-pyrazolo[3,4-f]isoquinoline-3-carboxylicacid trifluoroacetate,2-(3-{[2-(3,4-dimethoxyphenyl)ethyl]amino}propyl)-4,5-dihydro-2H-pyrazolo[3,4-f]isoquinoline-3-carboxylicacid trifluoroacetate,2-(1-benzofuran-2-yl)-5,6,8,9,10,1,1-hexahydro-7H-[1,4]diazepino[1′,2′:1,5]pyrazolo[3,4-f]isoquinolin-7-one,2-(3-{[4-(2-aminoethyl)phenyl]amino}propyl)-4,5-dihydro-2H-pyrazolo[3,4-f]isoquinoline-3-carboxylicacid trifluoroacetate,2-(1-naphthyl)-5,6,9,10-tetrahydropyrazino[1′,2′:1,5]pyrazolo[3,4-f]isoquinolin-7(8H)-one,8-(3-aminopropyl)-5,6,8,9,10,11-hexahydro-7H-[1,4]diazepino[1′,2′:1,5]pyrazolo[3,4-f]isoquinolin-7-onedihydrochloride,2-anilino-5,6,9,10-tetrahydropyrazino[1′,2′:1,5]pyrazolo[3,4-f]isoquinolin-7(8H)-one,2-(3-aminopropyl)-8-[2-(trifluoromethyl)phenyl]-4,5-dihydro-2H-pyrazolo[3,4-f]isoquinoline-3-carboxylic acid trifluoroacetate,9-(azidomethyl)-5,6,9,10-tetrahydropyrazino[1′,2′:1,5]pyrazolo[3,4-f]isoquinolin-7(8H)-one,9-({[2-(4-chlorophenyl)ethyl]amino}methyl)-5,6,9,10-tetrahydropyrazino[1′,2′:1,5]pyrazolo[3,4-f]isoquinolin-7(8H)-one,2-phenyl-5,6,8,9,10,11-hexahydro-7H-[1,4]diazepino[1′,2′:1,5]pyrazolo[3,4-f]isoquinolin-7-one,2-[3-(pentylamino)propyl]-4,5-dihydro-2H-pyrazolo[3,4-f]isoquinoline-3-carboxylicacid trifluoroacetate,10-(2-aminoethyl)-5,6,9,10-tetrahydropyrazino[1′,2′:1,5]pyrazolo[3,4-f]isoquinolin-7(8H)-one,2-[3-(allylamino)propyl]-4,5-dihydro-2H-pyrazolo[3,4-f]isoquinoline-3-carboxylicacid trifluoroacetate,2-(4-aminobutyl)-4,5-dihydro-2H-pyrazolo[3,4-f]isoquinoline-3-carboxylicacid dihydrochloride,2-(3-{[2-(4-aminophenyl)ethyl]amino}propyl)-4,5-dihydro-2H-pyrazolo[3,4-f]isoquinoline-3-carboxylicacid trifluoroacetate,2-[(1E)-3,3-dimethylbut-1-enyl]-5,6,9,10-tetrahydropyrazino[1′,2′:1,5]pyrazolo[3,4-f]isoquinolin-7-(8H)-one-trifluoroacetate,10-(nitromethyl)-5,6,9,10-tetrahydropyrazino[1′,2′:1,5]pyrazolo[3,4-f]isoquinolin-7(8H)-one,3-carboxy-2-[3-(methylammonio)propyl]-4,5-dihydro-2H-pyrazolo[3,4-f]isoquinolin-7-iumdichloride,2-[3-({[(4-butoxyphenyl)amino]carbonyl}amino)propyl]-4,5-dihydro-2H-pyrazolo[3,4-f]isoquinoline-3-carboxylicacid,2-{3-[(2-pyridin-3-ylethyl)amino]propyl}-4,5-dihydro-2H-pyrazolo[3,4-f]isoquinoline-3-carboxylicacid trifluoroacetate,2-{3-[(2-pyridin-2-ylethyl)amino]propyl}-4,5-dihydro-2H-pyrazolo[3,4-f]isoquinoline-3-carboxylicacid trifluoroacetate,2-{3-[(cyclopropylmethyl)amino]propyl}-4,5-dihydro-2H-pyrazolo[3,4-f]isoquinoline-3-carboxylicacid trifluoroacetate,2-{3-[(2-thien-2-ylpropyl)amino]propyl}-4,5-dihydro-2H-pyrazolo[3,4-f]isoquinoline-3-carboxylicacid trifluoroacetate,2-methoxy-5,6,9,10-tetrahydropyrazino[1′,2′:1,5]pyrazolo[3,4-f]isoquinolin-7(8H)-one,2-[3-(dimethylamino)phenyl]-5,6,9,10-tetrahydropyrazino[1′,2′:1,5]pyrazolo[3,4-q]isoquinolin-7(8H)-one,2-(3-{[2-(1H-pyrrol-1-yl)ethyl]amino}propyl)-4,5-dihydro-2H-pyrazolo[3,4-f]isoquinoline-3-carboxylicacid trifluoroacetate,2-[3-(benzyloxy)propyl]-8-quinolin-3-yl-4,5-dihydro-2H-pyrazolo[3,4-f]isoquinoline-3-carboxylicacid,2-{3-[(4-butoxybenzyl)amino]propyl}-4,5-dihydro-2H-pyrazolo[3,4-f]isoquinoline-3-carboxylicacid trifluoroacetate,2-(1,3-benzodioxol-5-yl)-5-,6,9,10-tetrahydropyrazino[1′,2′:1,5]pyrazolo[3,4-f]isoquinolin-7(8H)-one,2-[(E)-2-(2-fluorophenyl)ethenyl]-5,6,9,10-tetrahydropyrazino[1′,2′:1,5]pyrazolo[3,4-q]isoquinolin-7(8H)-one,2-[(1E)-hex-1-enyl]-5,6,9,10-tetrahydropyrazino[1′,2′:1,5]pyrazolo[3,4-f]isoquinolin-7(8H)-onetrifluoroacetate,2-anilino-5,6,8,9,10,11-hexahydro-7H-[1,4]diazepino[1′,2′:1′,5]pyrazolo[3,4-f]isoquinolin-7-one,5,6,8,9,10,11-hexahydro-7H-[1,4]diazepino[1′,2′:1,5]pyrazolo[3,4-f]isoquinolin-7-one,2-chloro-5,6,9,10-tetrahydropyrazino[1′,2′:1,5]pyrazolo[3,4-f]isoquinolin-7(8H)-one,2-[(E)-2-(4-methoxyphenyl)ethenyl]-5,6,9,10-tetrahydropyrazino[1′,2′:1,5]pyrazolo[3,4-f]isoquinolin-7(8H)-one,2-(methylthio)-5,6,9,10-tetrahydropyrazino[11′,2′:1,5]pyrazolo[3,4-f]isoquinolin-7(8H)-one,2-{3-[(2-furylmethyl)amino]propyl}-4,5-dihydro-2H-pyrazolo[3,4-f]isoquinoline-3-carboxylicacid trifluoroacetate,2-azepan-1-yl-5,6,9,10-tetrahydropyrazino[1′,2′:1,5]pyrazolo[3,4-f]isoquinolin-7(8H)-onetrifluoroacetate, 2-(3,6-dihydropyridin-1(2H)-yl)-5,6,9,10-tetrahydropyrazino[1′,2′:1,5]pyrazolo[3,4-f]isoquinolin-7(8H)-onetrifluoroacetate,9-methyl-5,6,9,10-tetrahydropyrazino[1′,2′:1,5]pyrazolo[3,4-f]isoquinolin-7(8H)-one,2-(piperidin-3-ylmethyl)-4,5-dihydro-2H-pyrazolo[3,4-f]isoquinoline-3-carboxylicacid hydrochloride,9-(chloromethyl)-5,6,9,10-tetrahydropyrazino[11′,2′:1,5]pyrazolo[3,4-f]isoquinolin-7(8H)-one,2-[(4-methoxybenzyl)amino]-5,6,9,10-tetrahydropyrazino[1′,2′:1,5]pyrazolo[3,4-f]isoquinolin-7(8H)-onetrifluoroacetate,2-(3-([2-(1H-imidazol-4-yl)-ethyl]amino}propyl)-4,5-dihydro-2H-pyrazolo[3,4-f]isoquinoline-3-carboxylicacid trifluoroacetate,2-(benzylamino)-5,6,9,10-tetrahydropyrazino[1′,2′:1,5]pyrazolo[3,4-f]isoquinolin-7(8H)-onetrifluoroacetate,2-(methylthio)-5,6,8,9,10,11-hexahydro-7H-[1,4]diazepino[1′,2′:1,5]pyrazolo[3,4-f]isoquinolin-7-one,2-{3-[(2-chlorobenzyl)amino]propyl}-4,5-dihydro-2H-pyrazolo[3,4-f]isoquinoline-3-carboxylicacid trifluoroacetate,2-[3-(benzylamino)propyl]-4,5-dihydro-2H-pyrazolo[3,4-f]isoquinoline-3-carboxylicacid trifluoroacetate;2-[3-({[(4-methoxyphenyl)amino]carbonyl}amino)propyl]-4,5-dihydro-2H-pyrazolo[3,4-f]isoquinoline-3-carboxylicacid,2-{3-[(2-phenylethyl)amino]propyl}-4,5-dihydro-2H-pyrazolo[3,4-f]isoquinoline-3-carboxylicacid trifluoroacetate,2-{3-[(thien-2-ylmethyl)amino]propyl}-4,5-dihydro-2H-pyrazolo[3,4-f]isoquinoline-3-carboxylicacid trifluoroacetate,2-benzyl-5,6,9,10-tetrahydropyrazino[1′,2′:1,5]pyrazolo[3,4-f]isoquinolin-7(8H)-one,5,6,8,9,10,11-hexahydro-7H-[1,4]diazepino[1′,2′:1,5]pyrazolo[3,4-f]isoquinolin-7-one,2-{3-[(4-chlorobenzyl)amino]propyl}-4,5-dihydro-2H-pyrazolo[3,4-f]isoquinoline-3-carboxylicacid trifluoroacetate,2-{3-[(2-phenylpropyl)amino]propyl}-4,5-dihydro-2H-pyrazolo[3,4-f]isoquinoline-3-carboxylicacid trifluoroacetate,7-oxo-5,6,7,8,9,10-hexahydropyrazino[1′,2′:1,5]pyrazolo[3,4-f]isoquinoline-9-carboxamidetrifluoroacetate,2-(3-hydroxypropyl)-4,5-dihydro-2H-pyrazolo[3,4-f]isoquinoline-3-carboxylicacid,2-(1,3-dihydro-2H-isoindol-2-yl)-5,6,9,10-tetrahydropyrazino[1′,2′:1,5]pyrazolo[3,4-f]isoquinolin-7(8H)-onetrifluoroacetate,2-{3-[(4-aminophenyl)amino]propyl}-4,5-dihydro-2H-pyrazolo[3,4-f]isoquinoline-3-carboxylicacid trifluoroacetate,2-(4-hydroxypiperidin-1-yl)-5,6,9,10-tetrahydropyrazino[1′,2′:1,5]pyrazolo[3,4-f]isoquinolin-7(8H)-onetrifluoroacetate,2-(3-aminopropyl)-7-hydroxy-8-(3-nitrophenyl)-4,5-dihydro-2H-benzo[g]indazole-3-carboxylicacid trifluoroacetate,2-(2-aminoethyl)-7-hydroxy-8-(3-nitrophenyl)-4,5-dihydro-2H-benzo[g]indazole-3-carboxylicacid trifluoroacetate,3-hydroxy-2-(3-nitrophenyl)-5,6,8,9,10,11-hexahydro-7H-benzo[g][1,4]diazepino[1,2-b]indazol-7-onetrifluoroacetate,2-(3-aminopropyl)-7-hydroxy-4,5-dihydro-2H-benzo[g]indazole-3-carboxylicacid dihydrochloride,2-(2-aminoethyl)-8-bromo-7-hydroxy-4,5-dihydro-2H-benzo[g]indazole-3-carboxylicacid trifluoroacetate,2-(3-aminopropyl)-8-[2-(4-chlorophenyl)ethyl]-7-hydroxy-4,5-dihydro-2H-benzo[g]indazole-3-carboxylicacid trifluoroacetate,3-hydroxy-2-(3-nitrophenyl)-5,6,9,10-tetrahydrobenzo[g]pyrazino[1,2-b]indazol-7(8H)-onehydrobromide,2-(3-aminopropyl)-8-bromo-7-hydroxy-4,5-dihydro-2H-benzo[g]indazole-3-carboxylicacid hydrobromide,2-(3-aminopropyl)-7-hydroxy-8-(4-nitrophenyl)-4,5-dihydro-2H-benzo[g]indazole-3-carboxylicacid trifluoroacetate,2-(3-aminopropyl)-8-(3-cyanophenyl)-7-hydroxy-4,5-dihydro-2H-benzo[g]indazole-3-carboxylicacid trifluoroacetate,2-(3-aminopropyl)-7-hydroxy-8-[3-(trifluoromethyl)phenyl]-4,5-dihydro-2H-benzo[g]indazole-3-carboxylicacid trifluoroacetate, and2-(3-aminopropyl)-7-hydroxy-8-(3,3,3-trifluoropropyl)-4,5-dihydro-2H-benzo[g]indazole-3-carboxylicacid trifluoroacetate,7-hydroxy-3,4,5,10-tetrahydro-1H-2,5-methanoazepino[3,4-b]indole-1-carboxylicacid,2,3,8,10,11,12-hexahydro-1H,7H-9,12-methanoazepino[3,4-b]pyrano[3,2-e]indole-8-carboxylicacid,7-methoxy-3,4,5,10-tetrahydro-1H-2,5-methanoazepino[3,4-b]indole-1-carboxylicacid,7-(methylthio)-3,4,5,10-tetrahydro-1H-2,5-methanoazepino[3,4-b]indole-1-carboxylicacid,7-(benzyloxy)-3,4,5,10-tetrahydro-1H-2,5-methanoazepino[3,4-b]indole-1-carboxylicacid,7-(methylthio)-3,4,5,10-tetrahydro-1H-2,5-methanoazepino[3,4-b]indole-1-carboxylicacid, 2,2,2-trifluoroethyl7-methoxy-3,4,5,10-tetrahydro-1H-2,5-methanoazepino[3,4-b]indole-1-carboxylate,2,3-dihydroxypropyl7-methoxy-3,4,5,10-tetrahydro-1H-2,5-methanoazepino[3,4-b]indole-1-carboxylate,pyridin-4-ylmethyl7-methoxy-3,4,5,10-tetrahydro-1H-2,5-methanoazepino[3,4-b]indole-1-carboxylate,2-fluoroethyl7-methoxy-3,4,5,10-tetrahydro-1H-2,5-methanoazepino[3,4-b]indole-1-carboxylate,allyl7-methoxy-3,4,5,10-tetrahydro-1H-2,5-methanoazepino[3,4-b]indole-1-carboxylate,benzyl7-methoxy-3,4,5,10-tetrahydro-1H-2,5-methanoazepino[3,4-b]indole-1-carboxylate,2-(methylthio)ethyl7-methoxy-3,4,5,10-tetrahydro-1H-2,5-methanoazepino[3,4-b]indole-1-carboxylate,2-methoxyethyl7-methoxy-3,4,5,10-tetrahydro-1H-2,5-methanoazepino[3,4-b]indole-1-carboxylatem,7-methoxy-3,4,5,10-tetrahydro-1H-2,5-methanoazepino[3,4-b]indole-1-carboxylicacid,7-hydroxy-3,4,5,10-tetrahydro-1H-2,5-methanoazepino[3,4-b]indole-1-carboxylicacid,2,3,8,10,11,12-hexahydro-1H,7H-9,12-methanoazepino[3,4-b]pyrano[3,2-e]indole-8-carboxylicacid,7-methoxy-3,4,5,10-tetrahydro-1H-2,5-methanoazepino[3,4-b]indole-1-carboxylicacid,7-(methylthio)-3,4,5,10-tetrahydro-1H-2,5-methanoazepino[3,4-b]indole-1-carboxylicacid,7-methoxy-3,4,5,10-tetrahydro-1H-2,5-methanoazepino[3,4-b]indole-1-carboxylicacid,7-(benzyloxy)-3,4,5,10-tetrahydro-1H-2,5-methanoazepino[3,4-b]indole-1-carboxylicacid, 7-(methylthio)-3,4,5,10-tetrahydro-1H-2,5-methanoazepino[3,4-b]indole-1-carboxylic acid,6-methoxy-2,3,4,9-tetrahydro-1H-beta-carboline-1-carboxylic acid,6-(2-oxo-2-phenylethoxy)-2,3,4,9-tetrahydro-1H-beta-carboline-1-carboxylicacid,6-methoxy-2-methyl-2,3,4,9-tetrahydro-1H-beta-carboline-1-carboxylicacid, 2,2,2-trifluoroethyl7-methoxy-3,4,5,10-tetrahydro-1H-2,5-methanoazepino[3,4-b]indole-1-carboxylate,6-methoxy-2,3,4,9-tetrahydro-1H-beta-carboline-1-carboxylic acid,7-hydroxy-3,4,5,10-tetrahydro-1H-2,5-methanoazepino[3,4-b]indole-1-carboxylicacid,6-hydroxy-2-methyl-2,3,4,9-tetrahydro-1H-beta-carboline-1-carboxylicacid,7-methoxy-3,4,5,10-tetrahydro-1H-2,5-methanoazepino[3,4-b]indole-1-carboxylicacid,6-methoxy-2-methyl-2,3,4,9-tetrahydro-1H-beta-carboline-1-carboxylicacid, 2,3-dihydroxypropyl7-methoxy-3,4,5,10-tetrahydro-1H-2,5-methanoazepino[3,4-b]indole-1-carboxylate,4-ethyl-6-methoxy-2,3,4,9-tetrahydro-1H-beta-carboline-1-carboxylicacid,6-methoxy-4-methyl-2,3,4,9-tetrahydro-1H-beta-carboline-1-carboxylicacid,8,9,10,11-tetrahydro-7H-pyrido[3′,4′:4,5]pyrrolo[2,3-f]isoquinolin-7-onetrifluoroacetate,3-(aminomethyl)-6-methoxy-2,3,4,9-tetrahydro-1H-beta-carbolin-1-onetrifluoroacetate,3-(aminomethyl)-6-methoxy-2,3,4,9-tetrahydro-1H-beta-carbolin-1-onehydrochloride, 7-methoxy-3,4,5,10-tetrahydroazepino[3,4-b]indol-1(2H)-one, 6-methoxy-2,3,4,9-tetrahydro-1H-beta-carbolin-1-one,6-methoxy-2,9-dihydro-1H-beta-carbolin-1-one,6-hydroxy-2,3,4,9-tetrahydro-1H-beta-carbolin-1-one,8,9,10,11-tetrahydro-7H-pyrido[3′,4′:4,5]pyrrolo-[2,3-f]isoquinolin-7-one,3-(aminomethyl)-6-methoxy-2,3,4,9-tetrahydro-1H-beta-carbolin-1-one,3-(aminomethyl)-6-methoxy-2,3,4,9-tetrahydro-1H-beta-carbolin-1-one,6-methoxy-3-{3-[(2-phenylethyl)amino]propyl}-2,3,4,9-tetrahydro-1H-beta-carbolin-1-one,(1E)-6-methoxy-2,3,4,9-tetrahydro-1H-carbazol-1-one oxime,(1Z)-6-methoxy-2,3,4,9-tetrahydro-1H-carbazol-1-one oxime,6-methoxy-3-{3-[(3-phenylpropyl)amino]propyl}-2,3,4,9-tetrahydro-1H-beta-carbolin-1-one,methyl 1-oxo-2,3,4,5-tetrahydro-1H-pyrido[4,3-b]indole-6-carboxylate,3-(hydroxymethyl)-6-methoxy-2,3,4,9-tetrahydro-1H-beta-carbolin-1-one,3-(3-aminopropyl)-6-methoxy-2,3,4,9-tetrahydro-1H-beta-carbolin-1-one,3-(2-aminoethyl)-6-methoxy-2,3,4,9-tetrahydro-1H-beta-carbolin-1-one,ethyl 1-(hydroxyimino)-2,3,4,9-tetrahydro-1H-carbazole-6-carboxylate,2-methoxy-7,8,9,10-tetrahydrocyclohepta[b]indol-6(5H)-one oxime,3-(hydroxymethyl)-6-methoxy-2,3,4,9-tetrahydro-1H-beta-carbolin-1-one,3-(3-aminopropyl)-6-methoxy-2,3,4,9-tetrahydro-1H-beta-carbolin-1-one,3-(2-aminoethyl)-6-methoxy-2,3,4,9-tetrahydro-1H-beta-carbolin-1-one,ethyl 1-(hydroxyimino)-2,3,4,9-tetrahydro-1H-carbazole-6-carboxylate,2-methoxy-7,8,9,10-tetrahydrocyclohepta[b]indol-6(5H)-one oxime,3-[3-(benzylamino)propyl]-6-methoxy-2,3,4,9-tetrahydro-1H-beta-carbolin-1-one,6-methoxy-2,3,4,9-tetrahydro-1H-carbazol-1-one oxime,6-iodo-2,3,4,9-tetrahydro-1H-carbazol-1-one oxime,6-methoxy-2-methyl-2,3,4,9-tetrahydro-1H-carbazol-1-one oxime,3-(3-hydroxypropyl)-6-methoxy-2,3,4,9-tetrahydro-1H-beta-carbolin-1-one,ethyl 1-oxo-2,3,4,5-tetrahydro-1H-pyrido[4,3-b]indole-6-carboxylate,6-methoxy-2,3,4,9-tetrahydro-1H-beta-carboline-1-thione, methyl4-oxo-2,3,4,9-tetrahydro-1H-carbazole-8-carboxylate, and2,3,4,9-tetrahydro-1H-carbazol-1-one oxime. Others are described in U.S.patent application Publication Nos. 2004-0127492, 2005-0101623,2005-0137220, and 2005-0143371.

Prodrugs and Other Modified Compounds

Interaction of a molecule, e.g., a drug, with a MAPKAP kinase-2polypeptide can be used to promote enhanced sensitivity of cells tochemotherapy or radiation treatment. The treatment, stabilization, orprevention of a disease or disorder associated with MAPKAP kinase-2 canbe mediated by administering a compound, peptide, or nucleic acidmolecule. In some cases, however, a compound that is effective in vitroin disrupting the interaction of a MAPKAP kinase-2 polypeptide and anatural substrate is not an effective therapeutic agent in vivo. Forexample, this could be due to low bioavailability of the compound. Oneway to circumvent this difficulty is to administer a modified drug, orprodrug, with improved bioavailability that converts naturally to theoriginal compound following administration. Such prodrugs may undergotransformation before exhibiting their full pharmacological effects.Prodrugs contain one or more specialized protective groups that arespecifically designed to alter or to eliminate undesirable properties inthe parent molecule. In one embodiment, a prodrug masks one or morecharged or hydrophobic groups of a parent molecule. Once administered, aprodrug is metabolized in vivo into an active compound.

Prodrugs may be useful for improving one or more of the followingcharacteristics of a drug: solubility, absorption, distribution,metabolization, excretion, site specificity, stability, patientacceptability, reduced toxicity, or problems of formulation. Forexample, an active compound may have poor oral bioavailability, but byattaching an appropriately-chosen covalent linkage that may bemetabolized in the body, oral bioavailability may improve sufficientlyto enable the prodrug to be administered orally without adverselyaffecting the parent compound's activity within the body.

A prodrug may be carrier-linked, meaning that it contains a group suchas an ester that can be removed enzymatically. Optimally, the additionalchemical group has little or no pharmacologic activity, and the bondconnecting this group to the parent compound is labile to allow forefficient in vivo activation. Such a carrier group may be linkeddirectly to the parent compound (bipartate), or it may be bonded via alinker region (tripartate). Common examples of chemical groups attachedto parent compounds to form prodrugs include esters, methyl esters,sulfates, sulfonates, phosphates, alcohols, amides, imines, phenylcarbamates, and carbonyls.

As one example, methylprednisolone is a poorly water-solublecorticosteroid drug. In order to be useful for aqueous injection orophthalmic administration, this drug must be converted into a prodrug ofenhanced solubility. Methylprednisolone sodium succinate ester is muchmore soluble than the parent compound, and it is rapidly and extensivelyhydrolysed in vivo by cholinesterases to free methylprednisolone.

Caged compounds may also be used as prodrugs. A caged compound may have,e.g., one or more photolyzable chemical groups attached that renders thecompound biologically inactive. In this example, flash photolysisreleases the caging group (and activates the compound) in a spatially ortemporally controlled manner. Caged compounds may be made or designed byany method known to those of skill in the art.

For further description of the design and use of prodrugs, see Testa andMayer, Hydrolysis in Drug and Prodrug Metabolism: Chemistry,Biochemistry and Enzymology, published by Vch. Verlagsgesellschaft Mbh.(2003). Other modified compounds are also possible in the methods of theinvention. For example, a modified compound need not be metabolized toform a parent molecule. Rather, in some embodiments, a compound maycontain a non-removable moiety that, e.g., increases bioavailabilitywithout substantially diminishing the activity of the parent molecule.Such a moiety could, for example, be covalently-linked to the parentmolecule and could be capable of translocating across a biologicalmembrane such as a cell membrane, in order to enhance cellular uptake.Exemplary moieties include peptides, e.g., penetratin or TAT. Anexemplary penetratin-containing compound according to the invention is,e.g., a peptide comprising the sixteen amino acid sequence from thehomeodomain of the Antennapedia protein (Derossi et al., J. Biol. Chem.269:10444-10450, 1994), particularly a peptide having the amino acidsequence RQIKIWFQNRRMKWKK (SEQ ID NO: 50), or including a peptidesequence disclosed by Lin et al. (J. Biol. Chem. 270:14255-14258, 1995).Others are described in U.S. patent application Publication No.2004-0209797 and U.S. Pat. Nos. 5,804,604, 5,747,641, 5,674,980,5,670,617, and 5,652,122. In addition, a compound of the invention couldbe attached, for example, to a solid support.

Screening Assays

Fluorescence polarization assays can be used in displacement assays toidentify small molecule peptidomimetics or other compounds useful in themethods of the invention. The following is an exemplary method for useof fluorescence polarization, and should not be viewed as limiting inany way. For screening, all reagents are diluted at the appropriateconcentration and the working solution, kept on ice. The working stockconcentration for GST and GST fusion proteins are ˜4 ng/μL,Fluorescein-labeled peptides can be used at a concentration of 1.56fmol/μL, while cold peptides at 25 pmol/μL. Samples are incubated at atotal volume of 200 μL per well in black flat bottom plates, Biocoat,#359135 low binding (BD BioSciences; Bedford, Mass.). Assays are startedwith the successive addition using a Labsystem Multi-prop 96/384 device(Labsystem; Franklin, Mass.) of 50 μL test compounds, diluted in 10%DMSO (average concentration of 28 μM), 50 μL of 50 mM MES-pH 6.5, 50 μLof Fluorescein-peptide, 50 μL of GST-MAPKAP kinase-2 polypeptide, or 50μL of unlabeled polypeptide can be used as a negative control. Onceadded, all the plates are placed at 4° C. Following overnight incubationat 4° C., the fluorescence polarization is measured using a Polarionplate reader (Tecan, Research Triangle Park, N.C.). A xenon flash lampequipped with an excitation filter of 485 nm and an emission filter of535 nm. The number of flashes is set at 30. Raw data can then beconverted into a percentage of total interaction(s). All furtheranalysis can be performed using SPOTFIRE data analysis software(SPOTFIRE, Somerville, Mass.)

Upon selection of active compounds, auto-fluorescence of the hits ismeasured as well as the fluorescein quenching effect, where ameasurement of 2,000 or more units indicates auto-fluorescence, while ameasurement of 50 units indicates a quenching effect. Confirmed hits canthen be analyzed in dose-response curves (IC₅₀) for reconfirmation. Besthits in dose-response curves can then be assessed by isothermaltitration calorimetry using a GST-MAPKAP kinase-2 polypeptide fusion.

Assays with a candidate compound may be performed in the presence of acompound known to bind MAPKAP kinase-2, and the difference in bindingthe presence and absence of the compound known to bind may be a usefulmeasure of the candidate compound's ability to bind to MAPKAP kinase-2.This assay may be done in any format known to those of skill in the art,e.g., as a displacement assay.

Alternate Binding and Displacement Assays

Fluorescence polarization assays are but one means to measurecompound-protein interactions in a screening strategy. Alternate methodsfor measuring compound-protein interactions are known to the skilledartisan. Such methods include, but are not limited to mass spectrometry(Nelson and Krone, J. Mol. Recognit., 12:77-93, 1999), surface plasmonresonance (Spiga et al., FEBS Lett., 511:33-35, 2002; Rich and Mizka, J.Mol. Recognit., 14:223-8, 2001; Abrantes et al., Anal. Chem.,73:2828-35, 2001), fluorescence resonance energy transfer (FRET) (Baderet al., J. Biomol. Screen, 6:255-64, 2001; Song et al., Anal. Biochem.291:133-41, 2001; Brockhoff et al., Cytometry, 44:338-48, 2001),bioluminescence resonance energy transfer (BRET) (Angers et al., Proc.Natl. Acad. Sci. USA, 97:3684-9, 2000; Xu et al., Proc. Natl. Acad. Sci.USA, 96:151-6, 1999), fluorescence quenching (Engelborghs, Spectrochim.Acta A. Mol. Biomol. Spectrosc., 57:2255-70, 70; Geoghegan et al.,Bioconjug. Chem. 11:71-7, 2000), fluorescence activated cellscanning/sorting (Barth et al., J. Mol. Biol., 301:751-7, 2000), ELISA,and radioimmunoassay (RIA).

Pharmaceutical Compositions

The pharmaceutical compositions of the present invention are prepared ina manner known per se, for example by means of conventional dissolving,lyophilizing, mixing, granulating or confectioning processes. Methodswell known in the art for making compositions and formulations arefound, for example, in “Remington: The Science and Practice ofPharmacy,” (20th ed., ed. A. R. Gennaro, 2000, Lippincott Williams &Wilkins, Philadelphia).

Solutions of the active ingredient, and also suspensions, and especiallyisotonic aqueous solutions or suspensions, are preferably used, it beingpossible, for example in the case of lyophilized compositions thatcomprise the active ingredient alone or together with a carrier, forexample mannitol, for such solutions or suspensions to be produced priorto use. The pharmaceutical compositions may be sterilized and/or maycomprise excipients, for example preservatives, stabilizers, wettingand/or emulsifying agents, solubilizers, salts for regulating theosmotic pressure and/or buffers, and are prepared in a manner known perse, for example by means of conventional dissolving or lyophilizingprocesses. The said solutions or suspensions may compriseviscosity-increasing substances, such as sodium carboxymethylcellulose,carboxymethylcellulose, dextran, poly vinylpyrrolidone or gelatin.

Suspensions in oil comprise as the oil component the vegetable,synthetic or semi-synthetic oils customary for injection purposes. Theremay be mentioned as such especially liquid fatty acid esters thatcontain as the acid component a long-chained fatty acid having from 8 to22, especially from 12 to 22, carbon atoms, for example lauric acid,tridecylic acid, myristic acid, pentadecylic acid, palmitic acid,margaric acid, stearic acid, arachidic acid, behenic acid orcorresponding unsaturated acids, for example oleic acid, elaidic acid,erucic acid, brasidic acid or linoleic acid, if desired with theaddition of anti oxidants, for example, vitamins E, β-carotene, or3,5-di-tert-butyl-4-hydroxytoluene. The alcohol component of those fattyacid esters has a maximum of 6 carbon atoms and is a mono- orpoly-hydroxy, for example a mono-, di- or tri-hydroxy, alcohol, forexample methanol, ethanol, propanol, butanol or pentanol or the isomersthereof, but especially glycol and glycerol. The following examples offatty acid esters are therefore to be mentioned: ethyl oleate, isopropylmyristate, isopropyl palmitate, “Labrafil M 2375” (poly oxyethyleneglycerol trioleate, Gattefoss, Paris), “Miglyol 812” (triglyceride ofsaturated fatty acids with a chain length of C₈ to C₁₂, Huls AG,Germany), but especially vegetable oils, such as cottonseed oil, almondoil, olive oil, castor oil, sesame oil, soybean oil and more especiallygroundnut oil.

The injection compositions are prepared in customary manner understerile conditions; the same applies also to introducing thecompositions into ampoules or vials and sealing the containers.

Pharmaceutical compositions for oral administration can be obtained bycombining the active ingredient with solid carriers, if desiredgranulating a resulting mixture, and processing the mixture, if desiredor necessary, after the addition of appropriate excipients, intotablets, drage cores or capsules. It is also possible for them to beincorporated into plastics carriers that allow the active ingredients todiffuse or be released in measured amounts.

Suitable carriers are especially fillers, such as sugars, for examplelactose, saccharose, mannitol or sorbitol, cellulose preparations and/orcalcium phosphates, for example tricalcium phosphate or calcium hydrogenphosphate, and binders, such as starch pastes using for example corn,wheat, rice or potato starch, gelatin, tragacanth, methylcellulose,hydroxypropylmethylcellulose, sodium carboxymethylcellulose and/orpolyvinyl-pyrrolidone, and/or, if desired, disintegrates, such as theabove-mentioned starches, also carboxymethyl starch, crosslinkedpolyvinylpyrrolidone, agar, alginic acid or a salt thereof, such assodium alginate. Excipients are especially flow conditioners andlubricants, for example silicic acid, talc, stearic acid or saltsthereof, such as magnesium or calcium stearate, and/or polyethyleneglycol. Drage cores are provided with suitable, optionally enteric,coatings, there being used, inter alia, concentrated sugar solutionswhich may comprise gum arabic, talc, polyvinylpyrrolidone, polyethyleneglycol and/or titanium dioxide, or coating solutions in suitable organicsolvents, or, for the preparation of enteric coatings, solutions ofsuitable cellulose preparations, such as ethylcellulose phthalate orhydroxypropylmethylcellulose phthalate. Capsules are dry-filled capsulesmade of gelatin and soft sealed capsules made of gelatin and aplasticiser, such as glycerol or sorbitol. The dry-filled capsules maycomprise the active ingredient in the form of granules, for example withfillers, such as lactose, binders, such as starches, and/or glidants,such as talc or magnesium stearate, and if desired with stabilisers. Insoft capsules the active ingredient is preferably dissolved or suspendedin suitable oily excipients, such as fatty oils, paraffin oil or liquidpolyethylene glycols, it being possible also for stabilisers and/orantibacterial agents to be added. Dyes or pigments may be added to thetablets or drage coatings or the capsule casings, for example foridentification purposes or to indicate different doses of activeingredient.

The pharmaceutical compositions comprise from approximately 1% toapproximately 95%, preferably from approximately 20% to approximately90%, active ingredient. Pharmaceutical compositions according to theinvention may be, for example, in unit dose form, such as in the form ofampoules, vials, suppositories, drages, tablets or capsules.

The formulations can be administered to human patients in atherapeutically effective amount (e.g., an amount that decreases,suppresses, attenuates, diminishes, arrests, or stabilizes thedevelopment or progression of a disease, disorder, or infection in aeukaryotic host organism). The preferred dosage of therapeutic agent tobe administered is likely to depend on such variables as the type andextent of the disorder, the overall health status of the particularpatient, the formulation of the compound excipients, and its route ofadministration.

For any of the methods of application described above, a compound thatinteracts with a MAPKAP kinase-2 polypeptide may be applied to the siteof the needed therapeutic event (for example, by injection, e.g., directinjection into one or more tumors), or to tissue in the vicinity of thepredicted therapeutic event or to a blood vessel supplying the cellspredicted to require enhanced therapy.

The dosages of compounds that interact with a MAPKAP kinase-2polypeptide depend on a number of factors, including the size and healthof the individual patient, but, generally, between 0.1 mg and 1000 mginclusive are administered per day to an adult in any pharmaceuticallyacceptable formulation. In addition, treatment by any of the approachesdescribed herein may be combined with more traditional therapies.

EXAMPLES Example 1 The p38MAPK-MK2 Pathway is Activated by Drugs thatDirectly Damage DNA

To investigate whether the p38 MAPK/MK2 pathway was involved in the DNAdamage response of cells following exposure to clinically usefulchemotherapeutic agents, we treated human U2OS osteosarcoma cells withthe DNA crosslinking agent cis-Platinum (cisplatin), the topoisomerase Iinhibitor camptothecin, or the topoisomerase II inhibitor doxorubicin(FIG. 12). p38 MAPK activation was assessed using an antibody specificfor the Thr-180/Tyr-182 doubly phosphorylated active form of the kinase.Activation of MK2 was monitored by its altered mobility on SDS-PAGE, andby immunoblotting using a phospho-specific antibody for pThr-344, a sitein the auto-inhibitory domain whose phosphorylation by p38MAPK resultsin a dramatic elevation of MK2 activity. Prior to exposure of cells toDNA damaging compounds (FIG. 12A-12C, zero hour lanes), or in cellstreated with DMSO (vehicle) alone (FIG. 12D), MK2 ran as a single bandthat did not cross-react with the anti-pThr-344 antibody. Within onehour after exposure of the cells to cisplatin and doxorubicin, or withinfour hour following treatment with camptothecin, MK2 displayed asignificant reduction in its electrophoretic mobility. The upshifted MK2band appeared with the same kinetics as both the MK2 pThr-344- and thep38MAPK pThr-180/pTyr-182 immunoreactive bands. Activation of MK2 wasentirely dependent on p38MAPK, since addition of the p38MAPK selectiveinhibitor SB203580 to the growth media 30 minutes prior to applicationof the DNA damaging agents completely abolished MK2 activation, whilepreserving activation of p38MAPK (FIG. 12E). Similar results for MK2activation in response to cisplatin, camptothecin and doxorubicin werealso observed in HeLa cervical carcinoma cells, U87MG human glioblastomacells, and primary MEFs (FIG. 29A and data not shown). The time courseof MK2 activation upon treatment with each of these drugs (FIG. 12A-12D)matched the rate of appearance of γ-H2AX nuclear foci (FIG. 12F). Thesedata indicate that treatment of cells with these genotoxic agentsresults in MK2 activation, likely as a direct result ofchemotherapy-induced DNA damage.

Example 2 ATM and ATR are Required for p38MAPK/MK2 Activation FollowingGenotoxin-Induced DNA Damage but not in Response to UV Irradiation

We analyzed the p38MAPK/MK2activation profile in ATM-deficient andATR-defective fibroblasts (FIGS. 13A and 13B, and FIG. 12G). We alsostudied the effect of pharmacological inhibition of these kinases byaddition of caffeine (FIG. 13C). Activation of the p38MAPK/MK2 complexin response to cisplatin, camptothecin or UV exposure occurred normallyin ATM deficient fibroblasts, while doxorubicin treatment failed toactivate either p38MAPK or MK2 in these cells. ATR-defectivefibroblasts, on the other hand, failed to activate p38MAPK or MK2following either cisplatin, doxorubicin, or camptothecin exposure.However, UV-induced p38MAPK/MK2 activation in these cells wasunaffected. Similarly, treatment of U2OS cells with 20 mM caffeine, aconcentration sufficient to inhibit ATM, ATR and DNA-PK, for thirtyminutes prior to exposure to cisplatin and doxorubicin completelyabrogated the p38MAPK/MK2 response, while the activation of thesekinases by UV occurred normally under these conditions. Taken together,these data indicate that cisplatin, camptothecin, and doxorubicinrequire ATR for p38MAPK/MK2 activation, that doxorubicin also requiresATM activity, and that UV irradiation is capable of activating thep38MAPK/MK2 in a manner that is independent of ATM, ATR, or DNA-PKfunction.

Example 3 Loss of p53 Renders Cells Dependent on MK2 Signaling forSurvival after Chemically-Induced DNA Damage

The p53 tumor suppressor protein plays an important role in the cellularresponse to DNA damage by transcriptionally upregulating the Cdkinhibitor p21 to induce a G₁ and G₂ arrest. Cancer cells frequently showdisruptions in the p53 pathway, eliminating this component of the DNAdamage response, and leaving the cells entirely dependent on remainingcheckpoint signaling pathways. To examine whether the ATR/ATMp38MAPK-MK2pathway was required for cell survival after genotoxin-induced DNAdamage in p53 wild-type and p53^(−/−) MEFs, we used RNAi to deplete MK2,and examined the response of these otherwise genetically identical cellsto cisplatin and doxorubicin using a colony survival assay (FIG. 29).Cells were infected with lentiviruses delivering shRNAs againstluciferase (control) or MK2 and analyzed 6 days later (FIG. 29C).Luciferase and MK2 knockdown MEFs were then exposed to increasing dosesof cisplatin or doxorubicin. As seen in FIGS. 29A and 29B, there was nodifference in the number of surviving colonies in the p53 wt/wt MEFs,regardless of the presence or absence of MK2, at any dose of cisplatinor doxorubicin examined. In contrast, downregulation of MK2 in p53^(−/−)cells dramatically reduced the number of surviving colonies. Theseresults demonstrate that depletion of MK2 specifically sensitizesp53-deficient cells to the anti-proliferative effects ofchemotherapy-induced DNA damage. We used Western blot analysis toprofile activation of the MK2 pathway and the p53 network followingcisplatin and doxorubicin treatment in these four cell lines (FIG. 29C).The presence or absence of MK2 had no effect on the strong induction ofp53 and p21 following exposure to cisplatin or doxorubicin in the p53WT/WT cells. Only minimal amounts of the p53 inducer protein p19ARF weredetected. Neither p53 or p21 induction was detectable in p53^(−/−) MEFsin the presence or absence of MK2. However, the tumor suppressor p19ARFwas strongly induced in these cells even in the absence of DNA damagingchemotherapy, likely reflecting a feedback response due to the inabilityof these cells to induce p53. Thus, we concluded that MK2 is notrequired for the normal p53/p21 induction or stabilization in responseto DNA damage in wild-type primary cells, and that 2 is unable to inducep21 expression after DNA damage in the absence of functional p53.

Example 4 Down-Regulation of MK2 Leads to Mitotic Catastrophe after DNADamage in p53^(−/−) Cells

We speculated that the reduced colony formation observed in MK2-depletedp53^(−/−) MEFs after DNA damaging chemotherapy might be due to mitoticcatastrophe resulting from defective cell cycle checkpoints. A hallmarkof mitotic catastrophe is entry of cells into mitosis despite thepresence of damaged DNA, resulting in activation of the apoptotic celldeath pathway. To investigate this, luciferase shRNA control and MK2depleted p53 wild-type or null MEFs were treated with low doses ofdoxorubicin or cisplatin for thirty hours and immunostained withantibodies against histone H3 pSer−10 as a marker for mitotic entry,histone γ-H2AX as marker for persistent DNA damage, and cleavedcaspase-3 as a marker for apoptosis (FIG. 30). Luciferase shRNA-treatedp53 wt/wt and p53^(−/−) cells showed robust γ-H2AX foci after exposureto DNA damaging chemotherapy. No phospho-histone H3 or cleaved caspase-3staining was observed in MK2-containing cells, consistent with an intactDNA damage response regardless of the presence (FIG. 30A) or absence(FIG. 30C) of p53. Similarly, an intact DNA damage checkpoint responsewas also observed in MK2 depleted cells that contained wild-type p53(FIG. 30B). In sharp contrast, however, in the MK2-deficient p53^(−/−)cells, a substantial fraction of the γ-H2AX positive cells also stainedpositively for both phospho-histone H3 and cleaved caspase 3 (FIG. 3D).Interestingly, no caspase 3 staining was observed in γ-H2AX-positivecells that did not also contain phospho-histone H3 (FIG. 30D,arrowhead). Thus, in the absence of M2, p53 null primary cells treatedwith cisplatin and doxorubicin lose one or more critical cell cyclecheckpoints and undergo mitotic catastrophe.

Example 5 Down-Regulation of MK2 Causes Regression of Establishedp53^(−/−) Tumors In Vivo after Low Dose Treatment with DNA DamagingAgents

We investigated whether the chemo-sensitizing effect of MK2 depletion inp53 null cells in culture could also be observed when pre-existing p53deficient tumors were treated with DNA damaging drugs in vivo. In theseexperiments HRas-V12 transformed p53^(−/−) MEFs were stably transfectedwith control shRNA or MK2 shRNA expressed from a murine U6 promoter,using a lentiviral delivering system (FIG. 18A). The lentiviral transfervector also encoded GFP under the control of a CMV promoter, allowingfor fluorescent detection of tumors in situ. Tumors were induced byinjection of 10⁶ cells into the flanks of nude mice. Twelve days later˜1 cm diameter tumors had formed at all injection sites, and treatmentwith cisplatin, doxorubicin, or vehicle was begun (FIG. 18B). In theabsence of treatment with DNA damaging drugs, the tumors arising fromthe MK2 depleted cells in the right flanks of these animals grewslightly larger than those of the luciferase shRNA control cells in theleft flanks (FIG. 18B-18D). Following treatment with cisplatin ordoxorubicin, the control tumors showed either minimal reduction in size,or slow continued growth (FIG. 18B, D, red symbols). In contrast, theMK2 depleted tumors showed a dramatic reduction in weight and diameter(FIG. 18B-18D, blue symbols). Tumors depleted of MK2 shrank from 1.3 cmto 0.4 cm over fourteen days when treated with cisplatin, and from 1.4to 0.5 cm when treated with doxorubicin. Thus, the sensitizing effect ofMK2 depletion on DNA damage induced cell death in p53-deficient primarycells observed in cell culture was also maintained in vivo. Theseresults strongly suggest that MK2 may be a useful target for the designof new cancer treatment agents.

Example 6 MK2 is Required for the G₂/M Checkpoint Following DoxorubicinTreatment in p53-Deficient Cells

To investigate the molecular mechanisms involved in MK2 dependentresponses to DNA lesions, we examined cell cycle profiles of control andMK2 depleted p53^(−/−) MEFs. Asynchronous MK2- or control knock-downp53^(−/−) MEFs were mock treated or exposed to doxorubicin for thirtyhour, and cell cycle distribution monitored by FACS. In one set ofexperiments the spindle poison nocodazole was added to the media threehours after addition of doxorubicin, to cause any cells progressingthrough the cell cycle to arrest in mitosis. DNA content was monitoredby PI staining; phospho-histone-H3 staining was used as an indicator ofmitotic entry. As shown in the left panels of FIG. 31A, treatment ofcontrol knock down p53^(−/−) cells with doxorubicin led to theaccumulation of cells with 4N DNA content, and a lack of phospho-histoneH3 staining in either the absence or presence of nocodazole, indicativeof an intact G₂/M checkpoint. These cells expressing control shRNAsbehaved identically to an untransfected control p53^(−/−) cellpopulation. In marked contrast, MK2 depleted p53^(−/−) cells treatedwith doxorubicin displayed a cell cycle profile similar to that ofuntreated cells (FIG. 31A, right upper and middle panels), with only asmall increase in the 4N peak compared to the doxorubicin-treatedluciferase shRNA controls, a slightly increased S-phase population, andthe appearance of a sub-G₁ population indicative of apoptosis. Additionof nocodazole following doxorubicin treatment to the MK2 depleted cellscaused them to accumulate in a 4N DNA containing peak, with 28.5% of thecells staining positively for phospho-histone H3 (FIG. 31A, right lowerpanels), a value similar to that of untreated p53^(−/−) cells blocked inmitosis with nocodazole. Intriguingly, MK2 depletion did not alter totalChk1 levels or reduce Chk1 activation following DNA damage (FIG. 31B).These findings demonstrate that loss of MK2 prevents p53-deficient cellsfrom establishing a functional G₂/M checkpoint followingdoxorubicin-induced DNA damage, despite the presence of activated Chk1.Identical results were obtained using a second unrelated shRNA againstMK2. Importantly, the checkpoint defect could be fully rescued in theMK2 depleted cells by expressing an shRNA-resistant form of MK2 atcomparable levels to the endogenous protein.

Two Cdc25 family members, Cdc25B and C, play important roles ininitiating and maintaining mitotic entry in normal cells, and areprominent targets of the G₂/M checkpoint. Cdc25B is believed to functionby activating Cdk1/Cyclin B at the centrosome in late G₂ as an initiatorof early mitotic events, while Cdc25C functions to further amplifyCdk1/CyclinB activity within a nuclear auto-amplification loop oncemitosis has begun. In response to γ- or UV-irradiation-induced DNAdamage, checkpoint kinases phosphorylate Cdc25B and C on Ser−323 and216, respectively, to induce their binding to 14-3-3 proteins,sequestering them in the cytoplasm away from their cyclin/Cdksubstrates. Cdc25B plays a particularly crucial role in initiating andmaintaining normal cell cycle G₂/M checkpoint responses, sincereactivation of Cdc25B is critical for DNA-damaged cells to re-enter thecell cycle. We therefore investigated whether MK2 signaling was requiredfor association of Cdc25B with 14-3-3 in response to DNA damage bychemotherapeutic drugs. As shown in FIG. 31C, both doxorubicin andcamptothecin treatment, resulted in the generation of stable14-3-3-binding sites on Cdc25B in the luciferase shRNA control cells. No14-3-3 binding of Cdc25B, however, was detected in lysates from the MK2depleted cells (FIG. 31C, lower panel). This result is in good agreementwith the cell cycle studies in panel A, which showed loss of the G₂/Mcheckpoint in MK2 depleted cells after treatment with the topoisomeraseinhibitor doxorubicin. These data indicate that loss of the G₂/Mcheckpoint after DNA lesions in MAPKAP Kinase-2-depleted p53-defectivecells likely arises, at least in part, from loss of Cdc25B binding to14-3-3 proteins.

Example 7 MK2 is Required for S-Phase Checkpoint Arrest FollowingCisplatin Treatment in p53-Deficient Cells

Treatment with the DNA intra-strand cross-linker cisplatin causedp53^(−/−) cells to predominantly accumulate in S phase of the cellcycle. RNA interference was used to investigate the role of MK2 in thisprocess. p53^(−/−) control knock-down cells showed an identicalaccumulation in S phase after cisplatin exposure (FIG. 32A, left middlepanels) as that seen in untransfected p53−/− cells. Addition ofnocodazole to the luciferase knock-down cells three hour followingcisplatin treatment did not reveal the appearance of any mitotic cellsover the ensuing twenty seven hours, as monitored by phospho-histone H3staining (FIG. 32A, lower left panels), indicating a functionally intactS-phase checkpoint. Depletion of MK2 prior to cisplatin exposureresulted in a dramatically different result. As seen in the right panelsof FIG. 32A, MK2-depleted p53^(−/−) cells showed a cell cycle profileafter cisplatin treatment that was similar to that of untreated cells,other than a slight increase in the total number of cells in S-phase andthe appearance of a sub-G₁ population consistent with apoptosis.Strikingly, when nocodazole was added three hours following cisplatinaddition, the MK2 depleted p53-r cells accumulated in a 4N DNAcontaining peak with ˜25% of the cells staining positive forphospho-histone H3. The same cell cycle defects after cisplatin exposurewere observed using a second unrelated shRNA sequence against M2, andthe MK2 shRNA phenotype was completely reversed by expression of anRNAi-resistant form of MK2 at physiological levels. Similar to what wasobserved following doxorubicin treatment (FIG. 31B), MK2 depletion didnot impair activation of Chk1 after cisplatin exposure (FIG. 32B). Thesedata imply that MK2 is essential for the cisplatin induced S-phasearrest in p53-deficient cells, and that loss of MK2 enables these cellsto override the cisplatin-induced S-phase checkpoint, despite thepresence of activated Chk1, and proceed into mitosis. In contrast to the14-3-3-mediated sequestration of Cdc25B and C involved in the G₂/Mcheckpoint response, the G₁ and S phase checkpoints are largelycontrolled by the phosphorylation-dependent degradation of another Cdc25isoform, Cdc25A. We therefore investigated whether MK2 was required forthe degradation of Cdc25A following cisplatin-induced DNA damage (FIG.32B). Cdc25A levels were undetectable in the control luciferaseknock-down cells after treatment with cisplatin. In contrast, in the MK2depleted cells, substantial amounts of Cdc25A remain present in thelysates after cisplatin exposure, indicating that in the absence ofMAPKAP Kinase-2, p53^(−/−) MEFs cells are defective in targeting Cdc25Afor degradation in response to cisplatin induced DNA damage. Thisimpaired ability of MK2 depleted cells to degrade Cdc25A likely explainstheir failure to establish a sustained G₁/S checkpoint followingcisplatin exposure. Interestingly, Cdc25A may be a direct target of MK2in vivo, since both MK2 and Chk1 phosphorylate Cdc25A equivalently invitro, Chk1 phosphorylation of Cdc25A in vivo has been shown tofacilitate its ubiquitin-mediated proteolysis in a complex andincompletely understood manner, and MK2 and Chk1 phosphorylate theidentical optimal sequence motifs when analyzed by oriented peptidelibrary screening.

Example 8 MK2 and Chk1 are Activated Independently by DNA Damage, andare Both Potently Inhibited by UCN-01

Activation of MK2 by cisplatin, camptothecin, and doxorubicin isstrikingly similar to the activation profile reported for Chk1.Similarly, the impaired S-phase and G₂/M checkpoints seen after theseDNA damaging stimuli in MK2 knock-down cells bears some resemblance towhat has been previously reported for Chk1-deficient p53-defectivecells. These phenotypic similarities prompted us to further investigatewhether the activation of Chk1 and MK2 was interdependent. As shown inFIGS. 31B and 32B, activation of Chk1 in response to doxorubicin andcisplatin was unimpaired in MK2 depleted cells. We thereforeinvestigated the converse—whether the activation of MK2 after DNA damagewas dependent on Chk1. U2OS cells depleted of Chk1 using siRNA wereexposed to cisplatin and doxorubicin, and analyzed for activation ofMK2. As shown in FIG. 19, phosphorylation/activation of MK2 occurrednormally after treatment with these DNA damaging agents, regardless ofthe presence or absence of Chk1. Thus, activation of MK2 and Chk1 afterdrug-induced DNA damage appears to occur independently of each other,and both kinases appear to participate in parallel DNA damage checkpointsignaling pathways that are necessary for cell survival in the absenceof a strong p53 response. The staurosporine derivative7-hydroxystaurosporin/UCN-01 has been shown to increase the cytotoxicityof chemotherapy and radiation and is currently in clinical trials. Ithas been demonstrated that a major target of UCN-01 is the checkpointkinase Chk1, leading to speculation that that the increased chemo- andradiation sensitivity of cells treated with UCN-01 is a direct result ofChk1-mediated checkpoint abrogation. UCN-01 inhibits Chk1 with an IC₅₀that is ˜1000 fold lower than that for Chk2, and hence has been usedexperimentally as a Chk1 specific inhibitor. Strong circumstantialevidence, however, suggests that UCN-01 must be inhibiting other kinasesinvolved in cell cycle control at similar concentrations as those usedfor Chk1 inhibition studies. For example, Chk1-depleted cells maintainphosphorylation of Ser−216, a well characterized Chk1 target site onCdc25C, both during asynchronous growth and following γ-irradiation.Phosphorylation at this site is lost, however, when cells are treatedwith low doses of UCN-01 (˜300 nM), indicating that UCN-01 inhibitablekinase(s) other than Chk1 participate in Cdc25C Ser−216 phosphorylation.Based on our finding that MK2 depletion results in a dramaticallyincreased chemosensitivity of malignant cells, we asked whether MK2might be a UCN-01 target, similar to Chk1. In vitro kinase assays wereperformed under identical reaction conditions with Chk1 and MK2 usingthe same optimal peptide substrate for both kinases with the coreconsensus sequence L-Q-R-Q-L-S-I, in the presence of variousconcentrations of UCN-01. As shown in FIG. 21A, UCN-01 potentlyinhibited both kinases, with an IC₅₀ value of ˜35 nM for Chk1 and ˜95 nMfor MAPKAP Kinase-2. The IC₅₀ value we measured for Chk1 is in goodagreement with previously published data. Importantly, the IC₅₀ value wemeasured for MK2 is significantly below the concentrations of UCN-01that are used in “Chk1-specific” checkpoint abrogation assays,suggesting that under the conditions used in those studies, both Chk1and MK2 were being inhibited. To examine the structural basis for UCN-01inhibition of MK2, the structure of the MK2:UCN-01 complex was modeledusing coordinates from the published MK2:staurosporine structure, andcompared the results with the co-crystal structure of Chk1:UCN-01 (FIG.21B). As seen in panels 2, 3 and 5, the 7-hydroxy moiety of UCN-01 canbe easily accommodated into the MK2:staurosporine structure, where itsclosest neighboring residues would be Val-118 (2.8 Å to Cγ2), Leu-141(3.2 Å to Cγ1), and Thr-206 (3.6 Å to Cγ2). Lack of steric hindrance,and the overall similarity of the modeled MK2:UCN-01 structure to theChk1:UCN-01 structure (panels 1, 4), provides a structural rationale forthe tight binding observed biochemically. To verify that MK2 is a directtarget of UCN-01 within cells, we measured the phosphorylation of theMK2 substrate hsp-27 after heat shock, a stimulus that activates thep38MAPK/MK2 pathway. Control or MK2 shRNA expressing U2OS cells wereincubated at 42° C. or 37° C. for two hours in the presence or absenceof 250 nM UCN-01, and phosphorylation of hsp-27 monitored byimmunoblotting. FIG. 21C shows that hsp27 is phosphorylated on Ser−82when the control luciferase shRNA cells were placed at 42° C. (lane 1).This phosphorylation was completely abrogated by treatment with UCN-01(lane 2). No phosphorylation was observed in MK2 knock-down cells placedat 42° C. regardless of the presence or absence of UCN-01 (lanes 3, 4),and no signal was observed in both the control and MK2 knock-down cellsthat were maintained at 37° C., with or without UCN-01 treatment (lane5-8). Heat shock was equally effective in promoting the phosphorylationof hsp-27 on Ser−82, and UCN-01 was equally effective in blocking Ser−82phosphorylation in cells that were depleted of Chk1 (FIG. 21D, lanes1-4). Thus, UCN-01 inhibits MK2 in vivo, and this effect is independentof Chk1 function. This data demonstrates that UCN-01 is a directinhibitor of MK2 within cells, and indicates that the clinical efficacyof UCN-01 in cancer treatment, particularly in p53-defective tumors,likely arises from the simultaneous inhibition of both the Chk1 and MK2signaling pathways

Example 9 A Model for Re-Wiring of Cell Cycle Checkpoint Pathways inp53-Proficient and Deficient Cells

Checkpoint function in p53-proficient cells is mediated primarilythrough a robust, sustained p53 response downstream of ATM, togetherwith Chk1 (FIG. 33A). Although not shown explicitly in the diagram, Chk1has been shown to also directly phosphorylate p53. Under theseconditions the presence of MK2 is not required for cell survival afterexposure to DNA damaging agents. In p53-deficient cancer cells,checkpoint signaling following exposure to DNA damaging agents ismediated through the combined action of both the Chk1 and the p38MAPK/MK2 pathways (FIG. 33B). In this situation the p38MAPK/MK2 branchof checkpoint signaling becomes essential for cell survival after DNAdamage. Both pathways are simultaneously inhibited by theindolocarbazole drug UCN-01.

Other Embodiments

All publications, patents, and patent applications mentioned in theabove specification are hereby incorporated by reference. In addition,U.S. patent application Publication Nos. US 2005-0196808 and US2006-0052951 are hereby incorporated by reference. Various modificationsand variations of the described method and system of the invention willbe apparent to those skilled in the art without departing from the scopeand spirit of the invention. Although the invention has been describedin connection with specific embodiments, it should be understood thatthe invention as claimed should not be unduly limited to such specificembodiments. Indeed, various modifications of the described modes forcarrying out the invention that are obvious to those skilled in the artare intended to be within the scope of the invention.

Other embodiments are in the claims.

1. A method for treating a cellular proliferative disorder in a patient, said method comprising the steps of: (a) determining whether said patient has a p53-deficient cell; and (b) if said patient has a p53-deficient cell, administering to said patient a compound that is capable of inhibiting an activity of a MAPKAP kinase-2 polypeptide.
 2. The method of claim 1, further comprising administering to said patient a chemotherapeutic agent, wherein said compound and said chemotherapeutic agent are administered in amounts sufficient to treat said cellular proliferative disorder in said patient, and wherein said chemotherapeutic agent is administered simultaneously or within twenty-eight days of administering said compound.
 3. The method of claim 2, wherein said chemotherapeutic agent is selected from the group consisting of alemtuzumab, altretamine, aminoglutethimide, amsacrine, anastrozole, azacitidine, bleomycin, bicalutamide, busulfan, capecitabine, carboplatin, carmustine, celecoxib, chlorambucil, 2-chlorodeoxyadenosine, cisplatin, colchicine, cyclophosphamide, cytarabine, cytoxan, dacarbazine, dactinomycin, daunorubicin, docetaxel, doxorubicin, epirubicin, estramustine phosphate, etodolac, etoposide, exemestane, floxuridine, fludarabine, 5-fluorouracil, flutamide, formestane, gemcitabine, gentuzumab, goserelin, hexamethylmelamine, hydroxyurea, hypericin, ifosfamide, imatinib, interferon, irinotecan, letrozole, leuporelin, lomustine, mechlorethamine, melphalen, mercaptopurine, 6-mercaptopurine, methotrexate, mitomycin, mitotane, mitoxantrone, nilutamide, nocodazole, paclitaxel, pentostatin, procarbazine, raltitrexed, rituximab, rofecoxib, streptozocin, tamoxifen, temozolomide, teniposide, 6-thioguanine, topotecan, toremofine, trastuzumab, vinblastine, vincristine, vindesine, and vinorelbine.
 4. The method of claim 1, said method further comprising radiation therapy, wherein said compound and said radiation therapy are administered in amounts sufficient to treat said cellular proliferative disorder in said patient, and wherein said radiation therapy is administered simultaneously or within twenty-eight days of administering said compound.
 5. The method of claim 1, wherein said cellular proliferative disorder is a neoplasm.
 6. The method of claim 5, wherein said neoplasm is cancer.
 7. The method of claim 6, wherein said cancer is selected from the group consisting of acoustic neuroma, acute leukemia, acute lymphocytic leukemia, acute monocytic leukemia, acute myeloblastic leukemia, acute myelocytic leukemia, acute myelomonocytic leukemia, acute promyelocytic leukemia, acute erythroleukemia, adenocarcinoma, angiosarcoma, astrocytoma, basal cell carcinoma, bile duct carcinoma, bladder carcinoma, brain cancer, breast cancer, bronchogenic carcinoma, cervical cancer, chondrosarcoma, chordoma, choriocarcinoma, chronic leukemia, chronic lymphocytic leukemia, chronic myelocytic leukemia, colon cancer, colon carcinoma, craniopharyngioma, cystadenocarcinoma, embryonal carcinoma, endotheliosarcoma, ependymoma, epithelial carcinoma, Ewing's tumor, glioma, heavy chain disease, hemangioblastoma, hepatoma, Hodgkin's disease, large cell carcinoma, leiomyosarcoma, liposarcoma, lung cancer, lung carcinoma, lymphangioendotheliosarcoma, lymphangiosarcoma, macroglobulinemia, medullary carcinoma, medulloblastoma, melanoma, meningioma, mesothelioma, myxosarcoma, neuroblastoma, non-Hodgkin's disease, oligodendroglioma, osteogenic sarcoma, ovarian cancer, pancreatic cancer, papillary adenocarcinomas, papillary carcinoma, pinealoma, polycythemia vera, prostate cancer, rhabdomyosarcoma, renal cell carcinoma, retinoblastoma, schwannoma, sebaceous gland carcinoma, seminoma, small cell lung carcinoma, squamous cell carcinoma, sweat gland carcinoma, synovioma, testicular cancer, uterine cancer, Waldenstrom's fibrosarcoma, and Wilm's tumor.
 8. The method of claim 1, said compound comprising UCN-01.
 9. The method of claim 1, said compound comprising a covalently-linked moiety capable of translocating across a biological membrane.
 10. The method of claim 9, said moiety comprising a penetratin peptide or TAT peptide.
 11. The method of claim 1, wherein said activity is substrate binding.
 12. The method of claim 1, wherein said compound is administered to said patient in the form of a prodrug.
 13. The method of claim 1, said compound comprising a double-stranded small interfering nucleic acid (siNA) molecule that is capable of directing cleavage of a MAPKAP kinase-2 RNA via RNA interference, wherein: (a) each strand of said siNA molecule is about 18 to 23 nucleotides in length; and (b) one strand of said siNA molecule comprises a nucleotide sequence that is substantially identical to the sequence of said MAPKAP kinase-2 RNA, wherein said siNA molecule is capable of directing cleavage of said MAPKAP kinase-2 RNA via RNA interference.
 14. The method of claim 13, wherein said siNA molecule comprises RNA.
 15. The method of claim 14, wherein the sequence of one strand of said siNA molecule comprises any one of SEQ ID NOs: 29-32.
 16. The method of claim 1, said compound comprising a nucleobase oligomer, wherein the sequence of said oligomer is complementary to at least 10 consecutive residues of a nucleotide sequence encoding a MAPKAP kinase-2 polypeptide.
 17. The method of claim 1, said compound comprising a peptide.
 18. The method of claim 17, said peptide comprising the amino acid sequence [L/F/I]XR[Q/S/T]L[S/T][Hydrophobic] (SEQ ID NO: 17), wherein said peptide comprises no more than 50 amino acids.
 19. The method of claim 18, said peptide comprising the amino acid sequence LQRQLSI (SEQ ID NO: 16).
 20. The method of claim 2, wherein said patient has a tumor, and wherein said compound is administered to said tumor.
 21. The method of claim 20, wherein said compound is administered to said tumor by direct injection.
 22. The method of claim 20, wherein said administration results in a reduction in size of said tumor, in comparison to a control patient to whom said compound is not administered.
 23. The method of claim 1, wherein said compound is capable of specifically inhibiting an activity of a MAPKAP kinase-2 polypeptide.
 24. The method of claim 1, wherein said administering induces increased sensitivity of said p53-deficient cell to a chemotherapeutic agent, in comparison to a p53-deficient control cell in a control patient to whom said compound is not administered.
 25. The method of claim 24, wherein said chemotherapeutic agent is selected from the group consisting of alemtuzumab, altretamine, aminoglutethimide, amsacrine, anastrozole, azacitidine, bleomycin, bicalutamide, busulfan, capecitabine, carboplatin, carmustine, celecoxib, chlorambucil, 2-chlorodeoxyadenosine, cisplatin, colchicine, cyclophosphamide, cytarabine, cytoxan, dacarbazine, dactinomycin, daunorubicin, docetaxel, doxorubicin, epirubicin, estramustine phosphate, etodolac, etoposide, exemestane, floxuridine, fludarabine, 5-fluorouracil, flutamide, formestane, gemcitabine, gentuzumab, goserelin, hexamethylmelamine, hydroxyurea, hypericin, ifosfamide, imatinib, interferon, irinotecan, letrozole, leuporelin, lomustine, mechlorethamine, melphalen, mercaptopurine, 6-mercaptopurine, methotrexate, mitomycin, mitotane, mitoxantrone, nilutamide, nocodazole, paclitaxel, pentostatin, procarbazine, raltitrexed, rituximab, rofecoxib, streptozocin, tamoxifen, temozolomide, teniposide, 6-thioguanine, topotecan, toremofine, trastuzumab, vinblastine, vincristine, vindesine, and vinorelbine.
 26. The method of claim 24, said compound comprising UCN-01.
 27. The method of claim 24, wherein said administering alters a DNA damage-responsive cell cycle checkpoint of said p53-deficient cell in comparison to said control cell.
 28. The method of claim 27, wherein said DNA damage-responsive cell cycle checkpoint is G₁/S phase arrest.
 29. The method of claim 28, wherein Cdc25a degradation in a p53-deficient cell is impaired in comparison to a p53-deficient control cell in a control patient.
 30. The method of claim 27, wherein said DNA damage-responsive cell cycle checkpoint is G₂/M phase arrest.
 31. The method of claim 30, wherein the interaction between Cdc25b and a 14-3-3 protein is reduced in comparison to a p53-deficient control cell in a control patient.
 32. The method of claim 27, wherein said cell cycle checkpoint alteration induces cell death.
 33. The method of claim 32, wherein said cell death is by apoptosis.
 34. The method of claim 33, wherein said apoptosis is at least partially dependent on caspase-3 activation. 